|Year : 2019 | Volume
| Issue : 1 | Page : 6-10
Microscopic evaluation of the effect of low-level laser therapy on platelet-rich fibrin: A light microscopic histological study
Anurag Bhatnagar, Mallanagouda B Patil, Shobha Prakash
Department of Periodontics, College of Dental Sciences, Davangere, Karnataka, India
|Date of Web Publication||24-Jun-2019|
Dr. Anurag Bhatnagar
Room No. 4, Department of Periodontics, College of Dental Sciences, Davangere - 577 004, Karnataka
Source of Support: None, Conflict of Interest: None
Background: This study evaluated and compared the concentration of platelets/leukocytes, fibrin matrix, and its distribution in nonlaser-treated platelet-rich fibrin (PRF) and laser-treated PRF. Recently, PRF has been used for regeneration of soft and hard tissue, in periodontics and oral surgery. Low-level laser therapy (LLLT) has been used for many therapeutic purpose of tissue healing. The use of PRF treated with LLLT has shown to enhance better healing potential. Materials and Methods: In this study, we have selected five healthy controls and collected 10 ml of blood from them. All blood samples were centrifuged, and from each blood sample, two PRF clots were formed. A total of ten samples obtained were divided into two groups. Each group had five samples. One group was without LLL treatment (Group I) and other was treated with LLL (Group II). All the ten samples were processed for light microscopic examination. Results: In both the groups, the PRF membrane showed a similar macroscopic structure and microscopic distribution. In the buffy coat region, the concentration of platelets–leukocyte aggregate was little higher in Group I. In Group II, the distribution pattern of fibrin clot was slightly different with that of Group I. Conclusion: The Choukroun's PRF concept leads to specific clot architecture with platelets and fibrin meshwork, especially in the buffy coat region. Changes in the PRF components with LLLT may have an affect the healing and regenerative of soft and hard tissues.
Keywords: Blood platelets, leukocytes, light microscopy, low-level laser, platelet-rich fibrin
|How to cite this article:|
Bhatnagar A, Patil MB, Prakash S. Microscopic evaluation of the effect of low-level laser therapy on platelet-rich fibrin: A light microscopic histological study. Indian J Oral Health Res 2019;5:6-10
|How to cite this URL:|
Bhatnagar A, Patil MB, Prakash S. Microscopic evaluation of the effect of low-level laser therapy on platelet-rich fibrin: A light microscopic histological study. Indian J Oral Health Res [serial online] 2019 [cited 2019 Jul 19];5:6-10. Available from: http://www.ijohr.org/text.asp?2019/5/1/6/261151
| Introduction|| |
Platelet concentrates and low-level laser therapy (LLLT) are recent treatment modalities in tissue regeneration and faster wound healing due to its biostimulatory effect on osteoblast, fibroblast, and blood vessels., Blood platelets are known to be extremely sensitive cells, and on their exposure to different stimuli (Laser) undergo rapid changes leading to their activation (platelet degranulation).
The platelet-rich fibrin (PRF) clot combines many healing and immunity promoters present in the blood. Most studies on platelet concentrates only highlight the platelet and growth factors (GFs), rarely assess leukocytes and almost never analyze fibrin architecture which directly influences the biomaterial outcome.
In available literature, only few studies have been conducted to know the architectural changes and cellular composition of PRF treated with or without LLLT. Therefore, our study is to examine the composition and architecture of the PRF clot and to point out the structural differences between laser-treated PRF and nonlaser-treated PRF.
| Materials and Methods|| |
This study is approval by the Institutional Ethics Committee in accordance with the Helsinki Declaration of 1975, as revised in 2000. All five individuals were selected and recruited from the Department of Periodontics at College of Dental Sciences, Davangere, Karnataka, India. Subjects included were healthy from age group of 18-55 years belonging to any gender. Any history of smoking, pregnancy, diabetes, cancer, hypertension, or under any medication were excluded from the study. Enrolled individuals were given instructions, and written informed consent was taken before the study.
Collection of blood and preparation of platelet-rich fibrin
From each individual, 20 ml blood was collected by venipuncture of the antecubital vein using a two 10 ml sterile syringe of 24-gauge needle. 10 ml blood was transferred into vacutainer tube contained anticoagulant dipotassium – ethylenediaminetetraacetic acid and was used to determine the baseline concentration value of platelets and leukocytes in whole blood. The remaining blood was transferred to sterile glass tube that has no anticoagulant. Immediately, the glass tubes were kept in the centrifuge machine (REMI C258 centrifuge) and centrifuged at 3000 rpm for 10 min. The contents of the centrifuged tube were removed using sterile tweezers. A yellowish upper layer (PRF) was separated from the lower red layer using scissors and was placed in a sterile cup. PRF clots were then divided into two groups. Group I (Control) without laser treatment and Group II (experimental) treated with low-level laser (LLL) using diode laser of wavelength 810 nm for 60s, in continuous noncontact mode at 0.1W with diffused irradiation (Picasso, AMD Laser, USA) [Figure 1].
|Figure 1: Irradiation of platelet-rich fibrin with diode laser in noncontact diffuse mode|
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Histological procedures for light microscopy
All PRF clot samples from Group I and Group II were dehydrated in increasing gradients of alcohol (60%, 80%, 100%, and 100%) for 1 h and then placed in two grades of xylene before embedding into paraffin. After complete dehydration and embedding, each clot was sectioned into 6-μm thick sections. Sectioning was done to the long axis of the clot including the buffy layer using soft tissue microtome. About 20 sections (Group I, Group II; 10 each) were stained using Hemalaun and Eosin following their specific protocols. The slides were then observed under light microscopy at ×10 and ×40.
Calculation of concentrates
The concentration of platelets and leukocytes in PRF was calculated for each group indirectly, by determining the numerical difference between the residual platelet/leukocyte concentration (of the remaining serum after removal of PRF) and the baseline platelet/leukocyte concentration for each specific study sample. In this study, platelet/leukocyte concentration analysis was carried out using an electronic automated cell counter (Medonic M32B, Boule Diagnostics, Sweden).
Data were collected and entered into a spreadsheet (Microsoft Office 2010 Excel, Microsoft Corporation, Washington). The results were compared and analyzed statistically using SPSSR Version 13 for Windows. P < 0.05 was considered as statistically significant.
| Results|| |
Immediately after centrifugation, PRF clot showed two main distinct macroscopic observations with naked eye, a yellow portion constituting the main body lying in above portion of the clot, and a red portion located at the end of the clot. A yellow-white layer (buffy coat) was observed between yellow and red portion.
Under light microscopy
Sections (20 slides) of buffy coat region after staining was analyzed under light microscope for the concentration and distribution pattern of platelets–leukocytes. Platelet aggregates appeared dark blue/violet whereas red blood cells (RBCs) and leukocyte cytoplasm were not easily detectable and were dark pink. The leukocyte nuclei stained dark blue with Hemalaun and it resembled platelet aggregates, making it difficult to distinguish between the two. With the hematoxylin and eosin staining, the fibrin matrix appeared homogeneous in light pink bands with irregular arrangement [Figure 2]a and [Figure 2]b.
|Figure 2: (a) Group I - hematoxylin and eosin staining of platelet-rich fibrin at ×10, (b) Group II - hematoxylin and eosin staining of PRF irradiated with low-level laser at ×10|
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Platelet–leukocyte aggregate distribution
The slides were analyzed by observing the violet spots in the different areas of the membrane at ×10. These violet spots represented platelet aggregates. Dense aggregate as dark violet spots of platelet was seen in nonlaser-treated PRF group, whereas the density of spots were slightly less in laser-treated PRF group. However, there was not much difference observed between the two groups with respect to platelet aggregate distribution. Both the groups shared a common distribution pattern in the buffy coat region, i.e., the platelet distribution was homogeneous throughout the clot width in the buffy coat. The Group II showed burnt areas with no platelet aggregates when compared to the surrounding tissue.
Beyond the buffy coat base, a thick fibrin strand was observed. The fibrin network appeared to be well organized. When observed under low magnification (x10) a gauze thread-like pattern of fibrin matrix was seen. Fibrin clot observed in both the groups provided a very compact matrix [Figure 2]a and [Figure 2]b. At a higher magnification (×40), fibrin was clearly organized in parallel strands that appeared very thick and dense in both groups [Figure 3]a. However, slight variation was seen in Group II as slight loose arrangement of the fibrins and areas of haphazard arrangement of fibrin around the spaces. The area around burnt region showed melted fibrin structures which could not be distinguished from neighboring structures [Figure 3]b.
|Figure 3: (a) Group I - hematoxylin and eosin staining of platelet-rich fibrin at ×40 showing distribution of fibrin clot, (b) Group II - hematoxylin and eosin staining of platelet-rich fibrin irradiated with low-level laser at ×40 showing fibrin clot distribution|
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Calculation of the platelets and leukocyte concentrates in platelet-rich fibrin
The mean distribution of the concentration of platelets and leukocytes of five samples is depicted in [Table 1] along with the baseline values.
|Table 1: Mean calculated platelet-rich fibrin platelet and leukocyte concentration (n=5)|
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The statistical analysis showed that the concentration of platelets was more in both the groups compared to baseline value with higher significance in group I (P < 0.001) while in Group II it was only significant (P < 0.05) [Table 2]. Furthermore, comparison of Group II to baseline showed more concentration of platelets in Group II, but it was not significant (P > 0.05).
|Table 2: Statistical analysis of groups and baseline values for platelet concentration|
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The leukocyte concentration was less when compared to baseline value for both groups and was highly significant (P < 0.001). However no significant difference (P > 0.05) was observed when both groups were compared [Table 3].
|Table 3: Statistical analysis of groups and baseline values for leukocyte concentration|
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| Discussion|| |
PRF has been used in various fields of medical and dental due to its organized fibrin matrix with platelet and leukocyte concentrates below the matrix, having ability to release GFs to promote wound healing, bone regeneration, graft stabilization, and hemostasis. Due to unique property of this biomaterial, it is useful as osteoconductive filling material during a sinus lift procedure, bone graft protection and remodeling, treatment of bony defects, and other applications in various fields.,
In recent years, lasers have been used to accelerate wound healing. LLLT has shown to cause vasoactive effect by supplying direct biostimulative light energy to the cells causing relaxation of smooth muscle associated with endothelium bringing vasodilation, thus allowing oxygen flow and immune cells into tissue contributing to accelerated healing.
Studies have shown that within the power density range of 0.45 to 60 J/cm2 of specific wavelength, LLLT can stimulate fibroblast proliferation. LLLT also have stimulating effects on bone cells and can accelerate the repair process of the bone. More importantly, it also shows the effect on cytoskeleton structures like collagen. Studies have suggested that LLLT can improve wound healing by modulating the rearrangement of collagen, enhances release of Growth Factors from fibroblast and stimulate cell proliferation.,
The effects of laser light on platelet activation and platelet clumping are nonlinear, showing activation effect and maximal alterations in platelet activation and reactivity at low and moderate applied laser light energies.
Some studies demonstrated LLLT induces platelet degranulation and the release of substances stored in the alpha granules. Therefore, it can be possible that the use of PRF treated with LLLT could help in increasing the concentration of GFs in the wound healing, thus accelerates healing process. This hypothesis is supported by Nagata et al. who conducted a study on animals to analyze the effect of LLL with or without PRF on healing of a periodontal defect, where he found that LLL/PRF shows a positive effect on healing. El-Hayes et al. showed LLLT having a positive local biostimulative effect in the early stage of healing with PRF in the treatment of intrabony periodontal defects and postulated that the combination of both LLLT and PRF as treatment modalities could induce bone formation in the bone defect more than that of LLLT or PRF alone. Pugliese et al. in their experimental animal study determined the effect of LLL on the tissue collagen and found increased deposit of collagen.
There are various researches going on to fulfill the criteria of accelerated wound healing and regeneration of lost tissues. PRF in today's era is a proven material that has regenerative potential and improve wound healing capacity.
In this study, PRF was prepared from collected blood and treated with LLL, examined microscopically to find any structural changes, and comparison was made between laser-treated and nonlaser-treated PRF. Suggested biostimulation effect of diode laser has been seen within the range between 0.001 and 10 J/cm2 as a therapeutic window; hence, diode laser was used for 60s, in continuous mode at 0.1 W with diffused irradiation.
The study showed that the distribution of platelets was not uniform and it was more concentrated in the buffy coat region in all the groups. The pattern of overall distribution of cells and fibers was different in both cases. The distribution of platelet-leukocyte aggregates in laser-treated group was slightly less observed with areas of burns causing cell destruction as compared to other group. Histologically, there was sparse collagen distribution in the fibrin matrix in LLLT group as compared to PRF group alone where thick interwoven fibrin matrix was observed under high power microscopy (×40). The nonlaser-treated group findings are not in accordance to Dohan Ehrenfest et al. study who suggested PRF as a highly organized three-dimensional matrix system. In the laser-treated group, a slight increase in intercollagen distance was seen, suggesting a greater channel for inflammatory cells to move around along with stem cells and GFs.
The results obtained after the calculation of the residual platelet and leukocyte concentration in PRF in both the groups showed that the concentration of platelets was more in nonlaser group than the laser group which further supports the histological finding. Although leukocytes were detected indirectly using blood analyzer in the clot, there was not much difference in concentration between the two groups.
The fact that laser irradiation can activate platelet membrane receptors- Glycoprotein IIb/IIIa complex that modulates platelets ability to respond to activating agents like silica, further supports our study for using LLL. In recent years, a large amount of studies have been devoted to identify and apply GFs for the regeneration of periodontal tissues. The biological idea behind this concept is interesting, but several unsolved issues remain as important challenges in this field.
In process of search, PRF and laser have shown a greater potential to meet this challenge. The cell composition of PRF/LLL group implies that the biomaterial is a blood-derived tissue which is stable. The effectiveness of platelet concentrates in promoting the healing depends on the platelet-released GFs that improve the vascularization, and as these aggregates are entrapped in organized fibrin network, it can be used to accelerate soft and hard tissue healing and regeneration.
Further, the study can be extended including a bigger sample size and using scanning electron microscope analysis for more descriptive analysis. This study gives an insight possible variation in the concentration and distribution of the PRF components treated with LLL. Hence, more studies are required to substantiate this novel biomaterial.
| Conclusion|| |
PRF is an excellent discovery of tissue engineering, and its combination with LLLT can enhance regeneration. The development of new therapies will enhance the healing potential in the tissues. Such platelet activation by LLL can be effectively used in tissue regeneration and healing process, but standardize protocol remains to be established. This study presents evidence in favor of the use of treated PRF with low-level irradiations in practice.
We would like to thank Dr. Priyanka Paul, biostatistician, for her efforts in completion of statistics.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Nagata MJ, de Campos N, Messora MR, Pola NM, Santinoni CS, Bomfim SR, et al.
Platelet-rich plasma, low-level laser therapy, or their combination promotes periodontal regeneration in fenestration defects: A preliminaryin vivo
study. J Periodontol 2014;85:770-8.
Stein E, Koehn J, Sutter W, Wendtlandt G, Wanschitz F, Thurnher D, et al.
Initial effects of low-level laser therapy on growth and differentiation of human osteoblast-like cells. Wien Klin Wochenschr 2008;120:112-7.
Gresner P, Watała C, Sikurová L. The effect of green laser light irradiation on whole blood platelets. J Photochem Photobiol B 2005;79:43-50.
Dohan Ehrenfest DM, Del Corso M, Diss A, Mouhyi J, Charrier JB. Three-dimensional architecture and cell composition of a Choukroun's platelet-rich fibrin clot and membrane. J Periodontol 2010;81:546-55.
Dohan Ehrenfest DM, Bielecki T, Jimbo R, Barbé G, Del Corso M, Inchingolo F, et al.
Do the fibrin architecture and leukocyte content influence the growth factor release of platelet concentrates? An evidence-based answer comparing a pure platelet-rich plasma (P-PRP) gel and a leukocyte- and platelet-rich fibrin (L-PRF). Curr Pharm Biotechnol 2012;13:1145-52.
Dohan DM, Choukroun J, Diss A, Dohan SL, Dohan AJ, Mouhyi J, et al.
Platelet-rich fibrin (PRF): A second-generation platelet concentrate. Part I: Technological concepts and evolution. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;101:e37-44.
Bancroft JD. Theory and Practical of Histological Techniques. Ch. 6. Philadelphia, PA: Churchill Livingstone; 1990. p. 89.
Naik B, Karunakar P, Jayadev M, Marshal VR. Role of platelet rich fibrin in wound healing: A critical review. J Conserv Dent 2013;16:284-93.
] [Full text]
Surendranath P, Radhika A. Low level laser therapy – A review. IOSR J Dent Med Sci 2013;12:56-9.
Olban M, Wachowicz B, Koter M, Bryszewska M. The biostimulatory effect of red laser irradiation on pig blood platelet function. Cell Biol Int 1998;22:245-8.
El-Hayes KA, Zaky AA, Ibrahim ZA, Allam GF, Allam MF. Usage of low level laser biostimulation and platelet rich fibrin in bone healing: Experimental study. Dent Med Probl 2016;53:338-44.
Pugliese LS, Medrado AP, Reis SR, Andrade Zde A. The influence of low-level laser therapy on biomodulation of collagen and elastic fibers. Pesqui Odontol Bras 2003;17:307-13.
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]