Effect of Calcium Silicate Cements on Vascular Endothelial Growth Factor Release from Platelet-rich Fibrin and its Architectural Changes

INTRODUCTION

Platelet-rich fibrin (PRF) first described by Choukroun et al.¹ is referred as the second-generation platelet concentrate. Activated platelets are rich in growth factors like platelet-derived growth factor, transforming growth factors (TGF-β), insulin-like growth factor, epithelial growth factor, and vascular endothelial growth factor (VEGF).² Evidence from both in vivo and in vitro studies have shown their stimulatory effects on the mesenchymal cells implicating a vital role in wound repair and tissue regeneration.³ Due to the osteogenic and regenerative potential of PRF, its use has been established for various procedures in dentistry including pulp capping, apexogenesis, post-extraction sockets, and periodontal surgeries.⁴ Revascularization procedure is frequently used in cases of immature necrotic teeth as an alternative treatment modality to conventional root canal treatment. It aims at creating aseptic root canal space using antibiotics, disinfecting solutions and induction of bleeding for blood clot formation that leads to homing of stem cells. This is followed by placement of a scaffold-like PRFm over the blood clot for effective packing of a biocompatible material to seal the blood clot. Mineral trioxide aggregate (MTA) has been the preferred material for sealing in such cases owing to its established biocompatibility and great sealing ability. Other popular restorative materials used in dentistry include various calcium silicate-based materials like Biodentine, calcium-enriched mixture, and Theracal-LC and all of them have been studied and proven to possess a stimulatory effect on the dental pulp with regard to the reparative dentin formation. These materials could also be used in conjunction with platelet concentrates for various endodontic procedures like apexogenesis and pulp capping.⁵ TheraCal LC (Bisco Inc., Schamburg, IL, USA) is a light-cured resin-modified calcium silicate material indicated for use in direct and indirect pulp therapy. The material shows superior biocompatibility and is reported to be less cytotoxic than other resin-based light-cured liners due to release of more calcium ions compared to others.⁶ Association of MTA, Biodentine, and TGF release has been reported in various studies.^7,8 However, there is not much evidence on the correlation of these endodontic materials with VEGF. Besides the inherent ability to induce growth factor release, pH is another factor that has been shown to influence the release of growth factor from a membrane.⁹

Therefore, this study intended to evaluate and compare the changes in the release of VEGF from PRF as well as the changes observed in the fibrin architecture of PRF under the influence of different calcium silicate cements; MTA, Biodentine, and Theracal.

MATERIALS AND METHODS

The study was conducted in ITS Dental College, Hospital and Research Center, Greater Noida, India. Ethical clearance was obtained from the institutional ethical committee (Ref. No. ITSDCGN/PRIN/L/2020/009). Eight healthy volunteers (American Society of Anesthesiologist physical status; ASA 1) were randomly selected. They were informed about the nature of the study and a written informed consent was obtained from them.

The study followed a double blinding protocol where the volunteers were unaware of the type of material their sample would be tested with. Two investigators (SS, Laboratory technician) analyzing the samples for VEGF samples using enzyme-linked immunosorbent assay (ELISA) and PRFm under scanning electron microscopy (SEM) were blinded to the group of materials. Investigators (BR, AM) manipulating and preparing the samples could not be blinded.

RESULTS

SEM ANALYSIS RESULTS

DISCUSSION

Vascular endothelial growth factor is the most potent angiogenic and vasculogenic factor involved in tertiary dentin formation. Vascular endothelial growth factor has also been identified to induce proliferation and differentiation of human pulp cells into odontoblasts.¹⁰ According to Matsushita et al., VEGF produced by human dental pulp cells acts in an autocrine manner and promotes chemotaxis, proliferation, and differentiation of cells. These facts suggest that VEGF may be a useful growth factor in the repair of damaged pulp and dentin.¹¹

According to Su et al.,⁴ a continuous and steady release of the growth factors from PRF is seen up to 300 minutes if used immediately after preparation within 1 hour. Dohan Ehrenfest et al.² in their study showed that a very high release of VEGF occurred during the first 4 hours post-preparation of PRF and continued slowly till the 7th day.

In our study, we observed that Theracal in conjunction with PRF released the highest VEGF at 1 hour at pH as high as 9, whereas PRF alone produced the minimum amount of VEGF at 1 hour at pH 7. On comparing the VEGF release by Biodentine group to MTA and Theracal group, higher amount of VEGF release was seen with Biodentine as compared to control. The release of VEGF in the control group of PRF was less at 1 and 5 hours.

In a recent study by Youssef et al.,¹² MTA, Biodentine, and Emdogain were seen to increase VEGF release even at 14th day after application as a pulp-capping agent as compared to calcium hydroxide which showed reduction in the VEGF release. Mullaguri et al.¹³ conducted a study on TGF and concluded that PRF layered with Biodentine released more amount of TGF-b1 from PRF. Another study by Paranjpe et al. evaluated the effect of MTA on direct contact with human dental pulp cells and showed that MTA was capable of upregulating the release of VEGF from the cells besides promoting odontoblastic activity.¹⁴

It has been established that the changes in the pH may influence the functioning and architectural integrity of the PRF membrane and the alterations in its thickness, length, porosity, and branching may be assessed to note the changes.¹⁵ Increased lateral aggregation of clots and thickening of fibers has been attributed to calcium ions acting upon fibrinogen.¹⁶ In this study, SEM analysis revealed that experimental groups of MTA, Biodentine, and Theracal had similar effects on the surface architecture of the PRF membrane. This could be because all these three materials belong to the calcium silicate group. It must be kept in mind that with materials having an acidic pH during fibrin polymerization, architectural changes like thickening and cloudiness may be seen which may cause complete disruption of the fibrin network and hence render them detrimental to be used with PRF.¹⁷ At higher magnification of 10,000×, our observations in the SEM analysis showed a comparable change in the architecture in all groups as compared to control suggesting that all calcium silicate cements might similarly affect fibrin integrity.

Various studies have shown dental pulp stimulatory effects of VEGF besides its ability to promote angiogenesis.¹⁸ The findings of this study might have a beneficial clinical implication as the results showed an increased release of VEGF from PRFm when layered with Theracal. PRFm used in procedures like revascularization and pulpotomy may therefore be layered over with these inductive dental materials to enhance the healing and regenerative outcome. This study also implicates a possible use of Theracal as a coronal sealing material following a regenerative procedure. However, the sealing and solubility properties must be weighed against the bioinductive properties. In a study by Alazrag et al., Theracal showed the least solubility at one week when compared with MTA and Biodentine. In the same study, it was also seen that a higher frequency of marginal gaps was seen with Theracal.¹⁹

Based on the findings of this study, it is observed that all calcium silicate cements provide a conducive environment for regenerative procedures when used with PRF.

Our study showed that the highest release of VEGF from PRF occurred in the theracal group suggesting that it could be an appropriate material used as a seal over scaffolds used in regenerative endodontic therapy and pulpotomy. Theracal was tested in this study because of its easy manipulation and dispensing in syringe form that could increase operators’ ease as well. However, a limitation here was the light-curable nature of the material theracal compared to the conventional settings in MTA and Biodentine.

Considering the limitations of this in vitro study with less sample size, further comprehensive in vivo research is therefore required to assess its clinical success and applicability.

CONCLUSION

Vascular endothelial growth factor release is seen more from PRFm in contact with Theracal along with considerable yet minimal disruption of fibrin architecture. Therefore, Theracal may be considered as an alternative dental material to be used in endodontics wherever PRF has indicated as well as a sealing material in regenerative procedures. However, future research with a large sample is required to validate the results further.

CLINICAL SIGNIFICANCE

The results show an increased release of VEGF from PRFm when layered with Theracal. PRFm used in procedures like revascularization and pulpotomy may therefore be layered with these dental materials to enhance the regeneration and create a biocompatible seal.

REFERENCES

1. Choukroun J, Adda F, Schoeffler C, et al. Une opportunité en paro-implantologie: le PRF. Implantodontie 2000;42:55–62.

2. Dohan Ehrenfest DM, de Peppo GM, Doglioli P, et al. Slow release of growth factors and thrombospondin-1 in Choukroun’s platelet-rich fibrin (PRF): a gold standard to achieve for all surgical platelet concentrates technologies. Growth Factors 2009;27(1):63–69. DOI: 10.1080/08977190802636713.

3. Toffler M, Toscano N, Holtzclaw D, et al. Introducing Choukroun’s platelet rich fibrin (PRF) to the reconstructive surgery milieu. J Implant Adv Clin Dent 2009;2586:2086.

4. Su CY, Kuo YP, Tseng YH, et al. In vitro release of growth factors from platelet-rich fibrin (PRF): a proposal to optimize the clinical applications of PRF. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009;108(1):56–61. DOI: 10.1016/j.tripleo.2009.02.004.

5. Gandolfi M, Siboni F, Polimeni A, et al. In vitro screening of the apatite-forming ability, biointeractivity and physical properties of a tricalcium silicate material for endodontics and restorative dentistry. Dent J 2013;1(4):41–60. DOI: 10.3390/dj1040041.

6. Bortoluzzi EA, Niu L, Palani CD, et al. Cytotoxicity and osteogenic potential of silicate calcium cements as potential protective materials for pulpal revascularization. Dent Mat 2015;31(12):1510–1522. DOI: 10.1016/j.dental.2015.09.020.

7. Wattanapakkavong K, Srisuwan T. Release of transforming growth factor beta 1 from human tooth dentin after application of either ProRoot MTA or biodentine as a coronal barrier. J Endod 2019;45(6):701–705. DOI: 10.1016/j.joen.2019.03.011.

8. Laurent P, Camps J, About I. BiodentineTM induces TGF‐β1 release from human pulp cells and early dental pulp mineralization. Int Endod J 2012;45(5):439–448. DOI: 10.1111/j.1365-2591.2011.01995.x.

9. Wahlstrom O, Linder C, Kal_en A, et al. Variation of pH in lysed platelet concentrates influence proliferation and alkaline phosphatase activity in human osteoblast-like cells. Platelets 2007;18(2):113–118. DOI: 10.1080/09537100600800537.

10. Zhang J, Liu X, Yu W, et al. Effects of human vascular endothelial growth factor on reparative dentin formation. Mol Med Rep 2016;13(1):705–712. DOI: 10.3892/mmr.2015.4608.

11. Matsushita K, Motani R, Sakutal T, et al. The role of vascular endothelial growth factor in human dental pulp cells: induction of chemotaxis, proliferation, and differentiation and activation of the AP-1-dependent signaling pathway. J Dent Res 2000;79(8):1596–1603. DOI: 10.1177/00220345000790081201.

12. Youssef AR, Emara R, Taher MM, et al. Effects of mineral trioxide aggregate, calcium hydroxide, biodentine and Emdogain on osteogenesis, odontogenesis, angiogenesis and cell viability of dental pulp stem cells. BMC Oral Health 2019;19(1):1–9. DOI: 10.1186/s12903-019-0827-0.

13. Mullaguri H, Suresh N, Surendran S, et al. Role of pH changes on transforming growth factor-β1 release and on the fibrin architecture of platelet-rich fibrin when layered with biodentine, glass ionomer cement and intermediate restorative material. J Endod 2016;42(5):766–770. DOI: 10.1016/j.joen.2016.02.009.

14. Paranjpe A, Smoot T, Zhang H, et al. Direct contact with mineral trioxide aggregate activates and differentiates human dental pulp cells. J Endod 2011;37:1691–1695.

15. Collen A, Koolwijk P, Kroon M, et al. Influence of fibrin structure on the formation and maintenance of capillary-like tubules by human microvascular endothelial cells. Angiogenesis 1998;2(2):153–165. DOI: 10.1023/A:1009240522808.

16. Wolberg AS. Thrombin generation and fibrin clot structure. Blood Rev 2007;21(3):131–142. DOI: 10.1016/j.blre.2006.11.001.

17. Joison AN, Gallo FS. Effect of time, pH, and doses to obtain in vitro efficient fibrinolysis with a new microplasmin: importance of results for future in vivo studies. Int J Appl Pharm Sci Res 2012;1:342–346.

18. Friedlander LT, Coates D, Seymour G, et al. Vascularity and VEGF/VEGFR2 signaling in the dentine-pulp complex of immature and mature permanent teeth. Eur Endod J 2018;3(3):153–159. DOI: 10.14744/eej.2018.07269.

19. Alazrag MA, Abu-Seida AM, El-Batouty KM, et al. Adaptability, solubility and biocompatibility of TheraCal LC compared with MTA-Angelus and biodentine as a furcation perforation repair material. Res Squ 2020. DOI: 10.21203/rs.3.rs-16588/v1.