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 Table of Contents  
ORIGINAL RESEARCH
Year : 2021  |  Volume : 13  |  Issue : 4  |  Page : 407-414

Comparison of surface microtopography and mechanodegradation characteristics of platelet-rich fibrin membranes using two different centrifugation protocols


1 Department of Periodontics, PMS College of Dental Science and Research, Thiruvanathapuram, Kerala, India
2 Department of Periodontics, Saveetha Dental College and Hospital, Saveetha Institute of Medical and Technical Sciences (Deemed to be University), Chennai, Tamil Nadu, India
3 Central Analytical Facility, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences &Technology, Poojapura, Thiruvanathapuram, Kerala, India
4 Department of Biomaterial Science and Technology, Sree Chitra Tirunal Institute for Medical Sciences &Technology, Poojapura, Thiruvanathapuram, Kerala, India

Date of Submission02-Mar-2021
Date of Decision23-Jun-2021
Date of Acceptance24-Jun-2021
Date of Web Publication19-Aug-2021

Correspondence Address:
Dr. P R Arunima
Department of Periodontics, PMS College of Dental Science and Research, Thiruvanathapuram, Kerala.
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JIOH.JIOH_47_21

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  Abstract 

Aim: To compare the surface microtopography and in vitro mechanodegradation characteristics of two different platelet-rich fibrin (PRF) preparation protocols, that is, leukocyte platelet-rich fibrin (L-PRF) and advanced platelet-rich fibrin (A-PRF). Materials and Methods: For the present ex vivo study, blood samples were obtained from 20 healthy volunteers, who were divided into two groups: group I (L-PRF) and group II (A-PRF). For each experiment, five samples each were used for obtaining L-PRF and A-PRF membranes. Surface microtopography was demonstrated by using light microscopic studies and scanning electron microscope (SEM) analysis. Mechanical characteristics were evaluated by using Universal Testing Machine and nano Triboindentor. Membrane degradation properties were studied by using the Bradford method. The data were expressed in mean and standard deviation (SPSS 20.0 version, USA). Intergroup statistical significance was assessed by using an unpaired t-test and defined significant as P < 0.05. Results: Light microscopic examinations and SEM analysis have demonstrated that the L-PRF membrane contained thick densely arranged fibers than the A-PRF membrane. Mechanical characteristics have revealed that the tensile strength was higher, and the breaking strain was lower in the L-PRF membranes as compared with A-PRF membranes. The degradation profile demonstrated that the A-PRF membrane takes a prolonged time for degradation as compared with L-PRF. Conclusion: The present study justifies that differences in the centrifugation protocol have an influence on the surface microtopography and mechanodegradation characteristics of the PRF membrane.

Keywords: Centrifugation Force, Periodontitis, Platelet-rich Fibrin, Regeneration


How to cite this article:
Arunima P R, Varghese SS, Paul W, Philipose Pamadykandathil L. Comparison of surface microtopography and mechanodegradation characteristics of platelet-rich fibrin membranes using two different centrifugation protocols. J Int Oral Health 2021;13:407-14

How to cite this URL:
Arunima P R, Varghese SS, Paul W, Philipose Pamadykandathil L. Comparison of surface microtopography and mechanodegradation characteristics of platelet-rich fibrin membranes using two different centrifugation protocols. J Int Oral Health [serial online] 2021 [cited 2022 Jan 29];13:407-14. Available from: https://www.jioh.org/text.asp?2021/13/4/407/324147




  Introduction Top


Periodontal disease is usually treated by using regenerative approaches that can successfully restore function and structure of the periodontal attachment apparatus, including both mineralized and soft tissues.[1] Major outcomes during the regeneration of periodontal tissue involve the hierarchical formation of interfaces with periodontal ligament orientation, tissue integration, and alveolar bone regeneration.[2] The complexity of periodontal tissues and their healing process poses a grave challenge in the restoration of normal functional tissues. Several researchers have studied platelet-rich hemoderivatives as a potential source of structural proteins and cytokines that are involved in periodontal wound healing.[3],[4],[5]

The most common platelet-rich hemoderivatives are platelet-rich plasma (PRP) and platelet-rich fibrin (PRF). PRP, the first-generation platelet concentrate, is well established with common factors, including double centrifugation and addition of bovine serum and anticoagulants.[3] A plethora of research have demonstrated PRP as a viable therapeutic adjunct for hard and soft tissue regeneration.[6],[7],[8],[9],[10] The PRP consists of platelets that secrete granule content, which includes various types of molecules involved in tissue regeneration, such as chemokines, growth factors, and cytokines.[6],[11] There are a few limitations with the PRP product, such as nonuniformity in the protocol for preparation of PRP and the possibility of occurrence of bleeding episodes and coagulopathies due to a cross-reaction between human factor Va and bovine thrombin.[9],[10]

Further research was focused on alternative clinically feasible strategies involving the development of a second-generation platelet concentrate, leukocyte- and platelet-rich fibrin (L-PRF). L-PRF is prepared by the centrifugation of peripheral blood in the absence of an anticoagulant. Centrifugation leads to the formation of three layers in the sample tube: red blood cells (RBCs) at the base, the L-PRF clot in the middle, and acellular plasma at the top. The middle L-PRF layer consists of leukocytes (±50% of initial blood), fibrin, and platelets (±95% of initial blood).[12] The L-PRF clot can be compressed to form a membrane of up to 1 mm thickness.[13] Under in vitro conditions, the integrity of the L-PRF membranes can be retained for more than seven days (more than 28 days under in vivo conditions). These membranes possess antibacterial properties due to the presence of leukocytes.[13],[14] Through its unique fibrin architecture, the broad cellular composition (mostly leukocytes) and myriad of bioactive factors make L-PRF a natural healing biomaterial that can be used for tissue engineering (leukocyte-driven tissue engineering).[15],[16],[17]

Recently, L-PRF has been reported as a living biomaterial that enhances periodontal tissue regeneration.[18] Nevertheless, standard protocols for regenerative procedures involving L-PRF are required to obtain optimal outcomes. Using histological analysis, Ghanaati et al. reported that, at higher speeds of centrifugation, most of the leukocytes collect at the bottom of PRF scaffolds; whereas at low speeds, the leukocytes are more evenly distributed within the scaffolds.[19] Further, lower centrifugation speed and longer centrifugation duration lead to an increase in the activity of gingival fibroblasts and the release of growth factors. A modification of the preparation protocol of the PRF clot by reducing the applied relative centrifugal force resulted in an improved preparation termed as Advanced-PRF (A-PRF).[19],[20] There is a lack of literature on whether centrifugation protocol influences the microtopography and mechanodegradation characteristics of PRF membranes. This study aimed at comparing and evaluating surface microtopography, mechanical and degradation characteristics of two different preparation protocols for standard PRF (L-PRF) (12-min centrifugation time and 2700rpm) and A-PRF (14-min centrifugation time and 1500rpm).


  Materials and Methods Top


Study setting and design

The type of study design was ex vivo conducted for a period of six months (July 2019–December 2019). The study design and consent forms for all procedures performed with the study subjects were approved by the institutional ethical committee for human subjects at Saveetha University, Chennai, India (IHC registration number: SDC/PhD/01) in accordance with the Helsinki declaration of 1975 as revised in 2008. Power analysis was performed to calculate the sample size necessary to detect significant differences with a power of 90% at a permissible (α) error of 5% using G-Power 3.1.3 software. Nine milliliters of blood were obtained from the antecubital vein of 20 systemically healthy volunteers aged between 25 and 35 years old.

For each experiment, five samples each were used for obtaining L-PRF and A-PRF membranes. The inclusion criteria were: systemically healthy volunteers who were non-smokers and not on any systemic medications. The exclusion criteria were: subjects suffering from systemic diseases, history of anticoagulant medication, or any other antibiotic intake in the past three months. Licensed and certified professionals performed all blood-drawing procedures.

PRF membrane preparation protocol

Venous blood was centrifuged immediately after collection in glass tubes (Microcentrifuge KW 80 Almicro Instruments, Haryana, India). Two different protocols were used to obtain PRF: centrifugation at 2700rpm for 12 min to obtain L-PRF clots[21] and centrifugation at 1500rpm for 14 min to obtain A-PRF clots.[19] The centrifuged tubes contained three layers with a bottom RBC layer, a top-most acellular plasma layer, and a middle layer of fibrin clot-containing platelets. The PRF clots obtained were collected, and RBC was removed with scissors without any PRF membrane damage. Next, the clots were placed in the PRF box (GDC instruments, Punjab, India) and compressed to form an L-PRF or A-PRF membrane.

Light microscopic examination

The PRF membranes (L-PRF and A-PRF) obtained were fixed in 10% formalin solution at room temperature for 24 h. The specimens were then embedded in paraffin, and 4-µm sections were obtained and stained using hematoxylin and eosin (H&E), Giemsa, and Masson-Goldner’s trichrome stains (Sigma-Aldrich St Louis, MO). The stained sections were observed under a light microscope at 40× magnification (Nikon Eclipse E600, Nikon, Tokyo, Japan).

SEM analysis

For scanning electron microscopy (SEM) evaluation, total of ten phosphate-buffered saline (PBS) washed PRF samples (n = 5 for L-PRF and A-PRF membranes, each) in each group were fixed for 20 min in 2.5% glutaraldehyde-containing PBS solution. The fixed samples were dehydrated using 50%, 70%, 80%, 90%, and 100% ethanol solutions for 5 min each. Next, the samples were dried using 100% hexamethyl disilazane (HMDS) (Sigma Aldrich) for 3 min, washed to remove excess HMDS, and aerated overnight. The samples were then mounted on stubs, sputter-coated with gold, and observed using an SEM (TM-1000; Hitachi, Tokyo, Japan) with an accelerating voltage of 20kV, as previously described.[22]

Tensile test

The mechanical properties of PRF membranes (n = 6 for L-PRF and A-PRF each) were assessed using a desktop universal testing machine (EZ test; Shimadzu, Kyoto, Japan). The samples were gripped at each end using clamps (using slip-proof rubber sheets to prevent slippage, with an initial gauge length of 10 mm), and tensile strain and strength at break were evaluated using a stress–strain plot.[22],[23]

Microhardness evaluation

Microhardness evaluation was done by surface indentation test with T1 950 Nano Triboindenter (T1 950 NanoTriboindenter; Industron Nanotechnology Pvt. Ltd., Technopark Campus Trivandrum, Kerala) at <1 µN load. Dynamic mechanical analysis technique was used to evaluate the mechanical properties of the sample. Load function was defined such that the probe oscillates at a different frequency between 210 and 300 Hz and at an amplitude of 15 μN during the hold segment. Large numbers of indentations in short periods, up to six per second, were obtained. Indents were performed in a user-defined grid. Between indentations, a piezo laterally translated the probe to a new indent location. A phase shift between load amplitude and displacement amplitude was measured using lock-in amplifier, which was used to calculate mechanical properties. A total of five readings from different locations for each membrane were recorded, and the mean value was computed.[22]

Degradation test

The amount of protein was estimated using the Bradford method using Bradford reagent (BioRad, Germany). The PRF membranes (n = 5 for L-PRF and A-PRF each) were immersed in 0.25% trypsin-ethylene diamine tetra acetic acid (EDTA) solution and incubated at 37°C in a CO2 incubator. At an interval of every 20 min, 50 µL of sample was collected for up to 3 h and subjected to Coomassie Brilliant Blue G250 assay. This assay was based on the principle that free protein molecules react with the dye molecules to form a complex that has higher extinction coefficient compared with the free dye molecules. Ten microliters of each sample was treated with 200 µL of diluted dye solution. The color was allowed to develop for 5–30 min. The absorbance was measured at 595nm. Standard solutions of bovine serum albumin were used to plot a standard graph. Protein concentration in the fractions was evaluated against the standard curve.[23] The results were expressed as mean and standard deviation.

Statistical analysis

The data were expressed in mean and standard deviation. Statistical Package for Social Sciences (SPSS 20.0 version, USA) was utilized. The data followed normal distribution. Unpaired t-test was applied to find the statistical significance between the group I and group II. The test was applied at 95% confidence interval with 5% of chance. In the comparison, P value less than 0.05 (P < 0.05) was considered statistically significant.


  Results Top


Histochemical analysis

With H&E staining, the fibrin matrix appeared pink, whereas the aggregated platelets were dark blue, purple. The cytoplasm of leukocytes is dark pink and not easily detectable. The nuclei of the leukocytes are visible in blue with hematoxylin staining and are not easily distinguishable from platelet aggregates. With the tri-color Masson (modified from Goldman), the aggregated platelets are still dark blue, but the leukocytes are easily identifiable because of their red color. The leukocytes are still difficult to highlight within platelet aggregates. However, the line of demarcation between platelet aggregates/leukocytes is obvious [Figure]s 1A and B, 2A and B, 3A and B.
Figures 1–3: (A) and (B) With light microscopic examination at 40× magnification using H&E, Giemsa, and Masson’s trichrome staining, the cells were distributed more toward the distal end in the L-PRF membrane and evenly throughout the A-PRF. The fibrin organization was dense with less interfibrous space in L-PRF, whereas it was loosely arranged with more interfibrous space in A-PRF

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L-PRF

The longitudinal section of the L-PRF membrane demonstrated thick, densely arranged fibers with a minimal interfibrous space. Histochemical analysis revealed cells distributed throughout the clot, with a higher density toward the distal end [Figures 1A, 2A, 3A].

A-PRF

The A-PRF membrane demonstrated thin, loosely arranged fibers with a more interfibrous space. Compared with L-PRF, the A-PRF membrane exhibited a more even distribution of cells throughout the clot. Owing to the membranes being shrunken and small, light microscopy did not allow a more detailed examination of the cell bodies in the clots [Figures 1B, 2B, 3B].

SEM analysis

The SEM evaluation revealed that the PRF membrane is composed of a dense network of thick fibrin fibers and thin fibrillae that give it a 3D appearance. The upper white portion of the membrane did not harbor cell bodies and, thus, had a clear appearance. On the other hand, the lower red portion of the membrane consisted of several platelets, some leukocytes, and fewer RBCs embedded inside the fibrous network. Platelets were either embedded within the fibrous network or visible as aggregates. In the L-PRF membrane, the thick fibrin fibers were strongly polymerized with the matrix [Figure 4]A and B. In contrast, in the A-PRF membrane, the thin fibrin fibers were mildly polymerized with the fibrin matrix [Figure 4]C and D. All the visible cell bodies were trapped between the fibrin matrix, and a few lymphocytes were localized on the textured surface in the form of activated lymphocytes for both types of membranes.
Figure 4: In the L-PRF membrane, the thick fibrin fibers were polymerized with fibrin matrix (A) and (B). In contrast, in the A-PRF membrane, the thin fibrin fibers were polymerized with fibrin matrix (C) and (D). All the visible cell bodies were trapped within the fibrin matrix, and a few lymphocytes were localized on the textured surface of both membranes in the form of activated lymphocytes

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Mechanical characteristics

Stress was calculated by dividing the force by the initial tissue cross-sectional area. “Tensile strain at break” is defined as tensile strain at the tensile stress at break, if it breaks without yielding. “Maximum tensile stress” sustained by the test specimen during a tensile test represents tensile strength. It was demonstrated that tensile strength is slightly higher in L-PRF samples as compared with A-PRF samples (L-PRF: 0.016 ± 0.003 MPa, A-PRF: 0.015 ± 0.004 MPa), and breaking strain was reduced in L-PRF (79.36% ± 7.00%) as compared with A-PRF samples (82.58% ± 5.71%). The microhardness was 11.05 ± 0.007 MPa for the L-PRF membrane and 10.98 ± 0.008 MPa for the A-PRF membrane. No statistically significant difference (P = 0.646 for microhardness; P = 0.875 for tensile strength; P = 0.773 for breaking strain) was found between the groups, as shown in [Table 1] and [Table 2] and [Graph 1].
Table 1: Comparison between mean tensile strength and breaking strain of L-PRF (group I) and A-PRF (group II)

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Table 2: Comparison between mean microhardness (MPa) of L-PRF (group I) and A-PRF (group II)

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Graph 1: Comparison of mean tensile strength and breaking strain between L-PRF (group I) and A-PRF (group II).

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Degradation characteristics

Degradation of the membrane was assessed in EDTA and trypsin-containing PBS solution (Bradford method), as shown in [Graph 2]. The A-PRF membranes degraded faster than the L-PRF membranes. A comparison of mean values between L-PRF and A-PRF at 20 min, 40 min, 60 min, and 120 min showed a significant difference in the degradation pattern, but it was not statistically significant (P > 0.05). A slight decrease in L-PRF degradation profile was noticed at the 80-min incubation time and was pronounced at 100-min, 120-min, and 180-min incubation time intervals. The comparison of mean values at 80 min, 100 min, 140 min, 160 min, and 180 min showed a significant difference (P < 0.05) between A-PRF and L-PRF.
Graph 2: Comparison of protein degradation of two groups between L-PRF (group I) and A-PRF (group II) at different absorbance

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  Discussion Top


Therapeutic use of the platelet-rich fibrin matrix (PRFM) constitutes a relatively new horizon in the arena of periodontal tissue engineering, and it has been a breakthrough in the acceleration of hard and soft tissue healing. The PRF could be formed as a membrane that is capable of covering and protecting the wound site and of entrapping active molecules and cells.[15],[16],[17] The use of PRF as a cover membrane permitted a rapid epithelization of the surface of the site and represented an effective barrier versus epithelial cell penetration inside the bone defect.[24] It is necessary to define and understand the architecture and composition of PRFs to ensure reproducibility and predictable tissue regeneration. The study aimed at comparing and demonstrating surface microtopography, mechanical and degradation properties of two PRF membranes generated using two most widely accepted standardized protocols of PRF clot preparation, that is, centrifugation at 2700rpm for 12 min for L-PRF and centrifugation at 1500rpm for 14 min for A-PRF. This study is the first of its kind where the surface microtopography of PRF membranes (L-PRF and A-PRF) prepared with two different protocols was studied using light microscopic examination and SEM analysis, followed by the evaluation of their mechanodegradation characteristics.

As per the light microscopic studies using H&E, Giemsa, and Masson’s staining, a section of L-PRF membrane demonstrated a dense fibrin organization with minimal interfibrous space and a higher density of cell bodies in the lower portion [Figures 1A], 2A, and 3A]. The A-PRF membrane showed less fibrin organization with more interfibrous space as compared with the L-PRF membrane. The cell bodies were more as compared with L-PRF and were distributed evenly throughout the entire prepared A-PRF membrane, focusing more on to the lower portion [[Figures 1B], 2B, and 3B]. Our results indicated that lower centrifugation speed and higher centrifugation time have made this difference in the distribution of cells in PRF samples, although this observation was not definitively proven.

The SEM analysis of previous studies has demonstrated a nonuniform distribution of leukocytes and platelets in PRF clots.[22 These cell bodies are generally located between the fibrin clot and the RBC layer after centrifugation],[ and they are visible as a buffy coating over the surface of the PRF clot. In case the membrane is to be used for surgical purposes],[ this buffy layer must be collected along with the clot. This makes it essential to collect a small portion of the RBC layer along with the PRF clot to ensure maximal collection of these cell bodies. The platelet distribution becomes increasingly scarce as we move away toward the lower portion in both L-PRF and A-PRF samples. The upper portion seems to be more homogenous with fibrin organization [Figure 4A]–[D].

The clotting and aggregation processes also modify the morphology of platelets.[25] Hence, the identification of nonactivated platelets (discoid bodies) was not possible but rather only a large aggregate of platelet-fibrin layers could be identified. Both L-PRF and A-PRF contained platelets in the form of single entities (bodies) as well as aggregates [Figure 4A]–[D]. However, L-PRF and A-PRF membranes demonstrated amorphous, globular, and irregular platelets. Except for the smaller plasma aggregate size of L-PRF, there were no differences in the morphology of platelets present in both the membranes. The SEM analysis revealed spherical leukocytes with irregular surfaces; besides, L-PRF had thicker diameter fibers and a more condensed matrix as compared with A-PRF [Figure 4A]–[D].

Regenerative membranes must have strong mechanical properties to retain blood clots and accelerate wound healing. Our study demonstrated that microhardness was slightly higher for the L-PRF membrane (11.05 ± 0.007 MPa) as compared with the A-PRF membrane (10.98 ± 0.008 MPa), and the same was observed for tensile strength (L-PRF: 0.016 ± 0.003 MPa, A-PRF: 0.015 ± 0.004 MPa). In comparison, reduced breaking strain was observed in L-PRF (79.36% ± 7.00%) as compared with A-PRF (82.58% ± 5.71%). No statistically significant difference (P = 0.646 for microhardness; P = 0.875 for tensile strength; and P = 0.773 for breaking strain) was found between the groups, as shown by [Tables 1] and [2] and [Graph 1]. This might be due to the differences in centrifugation protocol that have affected the polymerization phase of the prepared membranes. These data matched with the previously published literature. The mechanical properties were lower in terms of modulus of elasticity and hardness for L-PRF as compared with commercially available guided tissue regeneration membranes.[22] However, another study compared the mechanical characteristics of autologous PRFM and L-PRF and demonstrated that tensile strength and maximum tensile strain of the L-PRF membrane were significantly higher than that of the PRFM (P < 0.01). The PRFM was significantly stiffer than the L-PRF membrane (P < 0.01).[25] Previous studies have shown that the mechanical properties of the fibrin matrix is significantly affected by the mode of polymerization; during centrifugation, a slow and natural PRF polymerization occurs.[26] The activated platelets also stimulate the formation of thrombin and induce subsequent polymerization of fibrinogen to form fibrin molecules. The fibrin matrix becomes stronger with an increase in the concentration of fibrinogen.[27] It has been demonstrated that a high concentration of thrombin in PRF clots results in the generation of fibrin with a smaller diameter and that these smaller fibrin fibers tend to pack more tightly.[26],[28] Thus, in the present study, lower centrifugation speed and force led to inadequate separation of blood components and the generation of a smaller clot (A-PRF) with a weaker biological signature and fibrin polymerization as compared with L-PRF.

Evaluation of degradation time for the PRF membrane is important with respect to its clinical application in periodontal regenerative procedures. If the PRF membrane degrades faster, it leads to incomplete tissue regeneration. The results of our study demonstrated that there was a difference in the degradation profile of PRF membranes. The first 1-h degradation rate was found to be uniform and constant at the 20-min, 40-min, and 60-min time interval for both samples. A comparison between the mean values of both membranes at 20 min, 40 min, 60 min, and 120 min showed a significant difference in the degradation pattern, but it was not statistically significant (P > 0.05). A slight decrease in the L-PRF degradation profile was noticed at the 80-min incubation time and was pronounced at the 100-min, 120-min, and 180-min incubation time intervals. The comparison of mean values at 80 min, 100 min, 140 min, 160 min, and 180 min showed a significant difference (P < 0.05) between both membranes. The fiber thickness appeared to be less in A-PRF and could be substantially higher; it paved a role for faster degradation as compared with L-PRF.[19],[23] The differences in centrifugation protocols used for the generation of PRF membranes have influenced the degradation characteristics of the two prepared samples. Adequate blood component partitioning could not be achieved using low centrifugal speed and force, which led to a smaller clot (A-PRF) with a weaker biological signature compared with that of L-PRF. Thus, higher centrifugal forces of around 400× g (2700rpm, 12 min) are required to yield a PRF membrane with desirable mechanodegradation characteristics for its clinical application in the field of periodontal regeneration.

Limitations

The influence of differences in centrifugation protocol in the biological signature of different types of PRF membranes was not clearly demonstrated and has not been published scientifically up to now. All centrifuges have specific mechanical characteristics that differ significantly among the many possible available models. This fact might have influenced our results. So a need for standardization of centrifuge and centrifugation protocols is necessary to reduce the error in future research. In addition although within the frame of the power analysis to detect significant differences, the relatively small sample size might have masked significant outcomes. Further studies are needed to better elucidate all possible effects of the PRF membrane for clinical application.

Clinical relevance

The differences in the centrifugation protocol have an influence on the mechanodegradation characteristics of the PRF membrane. The PRF membranes with good tensile strength and prolonged degradation characteristics provide a more suitable support for regeneration; thus, it enables the clinician to choose the appropriate PRF membrane type depending on the purpose of regenerative surgery.


  Conclusion Top


This study demonstrated that centrifugation force and time had influenced the surface microtopography and the mechanodegradation properties of PRF membranes prepared with two different protocols. The use of a lower g force and speed (1500rpm, 14 min for A-PRF) did not seem enough for proper separation of the blood constituents, and it led to the preparation of a clot (A-PRF) of a much smaller size, a weaker biological signature as compared with L-PRF. It confirms the need for using forces around 400g (2700rpm, 12 min), to yield a PRF membrane with desirable mechanodegradation characteristics for its clinical application in the field of periodontal regeneration.

Acknowledgment

The authors are grateful to Sree Chitra Tirunal Institute for Medical Sciences and Technology Biomedical Technology Wing, Poojapura, Thiruvananthapuram, Kerala; Industron Nanotechnology Pvt. Ltd., Thiruvanathapuram, Kerala; and Biogenix Research Centre, Poojapura, Thiruvananthapuram, Kerala, for their support and encouragement to conduct this study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

Author contributions

Not applicable.

Ethical policy and institutional review board statement

The present study is ethically approved by the Institutional Review Board, Saveetha University, Chennai, India (IHC registration number: SDC/PhD/01) and all the experimental procedures were conducted according to the guidelines of the Declaration of Helsinki 1975 as revised in 2008.

Declaration of patient consent

Signed consent forms were obtained from all the participants before enrolling them in the study.

Data availability statement

Data will be provided on a valid request basis by contacting the corresponding author’s mail.



 
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