Evaluation of mechanical properties of platelet-rich fibrin membrane for implant surgery: An analysis in vitro.

The aim of this study was to evaluate and compare the mechanical resistance of platelet-rich fibrin (PRF) membrane when submitted to resistance traction on longitudinal axis. Blood collection of a healthy individual was collected with an amount of 300 mL and divided into 30 tubes containing 10 mL each one. The samples were divided into three groups, according to the g-force protocols: (1) F200 g: (2) F400 g; (3) F800 g. Membranes of each g-force group were divided into subgroups, according to waiting time after centrifugation: (T0) immediate use and (T30) use after 30 min. Considering these intervals of time, the concentrate was removed from tubes and inserted in a PRF metallic box to confection of the membrane. The PRF membranes were submitted to mechanical tension on a universal testing machine and obtained to a resistance force of each membrane. The centrifugation time showed no statistically significant difference for membrane resistance for any force applied (P > 0.05; Student's t-test). For T0 group results demonstrated no influence for membrane resistance (P = 0.357; Student's t-test), therefore T30 showed statistically significant difference (P = 0.040; Student's t-test) for membrane resistance for centrifugation forces applied, with highest value when applied greatest force. The findings suggest that the waiting time for centrifugation could be determined according to demand of application, and for immediate use of the membrane, the centrifugation did not influence the resistance, on the other hand, after 30 min, the application of higher force resulted in a membrane with considerable resistance. Copyright:

INTRODUCTION

The oral rehabilitation with dental implants can find problems due when appears bone resorption with lack of hard tissue around implants, demonstrating failures for support of osseointegrated implants.[12]

Several approaches through regenerative procedures with biomaterials can be used for bone and tissue regeneration, such as, synthetic membrane, grafts, bioactive growth factors, and enamel matrix derivatives are some materials that can be used for regeneration.[3] In addition, the use of autologous blood concentrated is an alternative to be considered.[4]

The membrane leukocytes platelet-rich fibrin (L-PRF) in polymerized and gelled form and has function of a three-dimensional framework. This concentrate is composed of platelets, leukocytes, platelet micro particles, glycoprotein, and several plasma proteins.[5] In addition, these concentrates has function to release growth factors such as, growth factor beta, platelet-derived growth factor and endothelial growth factor. The presence of these elements can benefit the healing process, angiogenesis and bone neo-formation in the surgical area to obtain bone regeneration.[67]

The concentrate is obtained after human blood centrifugation, without additives and this process can be performed through different forces and times; the literature demonstrated a wide variety of possibilities regarding forces and times for centrifugation.[8] The centrifugation processes can influence the characteristics of the membrane.[910] Ghanaati et al.[9] demonstrated that when membrane was harvested from the lowest centrifugation force showed as a porous fibrin and with density lower, however, a greater number of leukocytes and growth factors were found added on the clot.

Considering the variety of centrifugation protocols and their influence on the characteristics of the membranes obtained, the literature demonstrates that is necessary to evaluate the centrifugation protocol which can influence directly in the resistance of membrane according to traction.[8910]

Otherwise, considering the clinical application, to obtain the membrane can be performed at the beginning of the surgical procedure or immediately before the use, it means, during the surgical procedure, interfering in the fibrin polymerization time.

The aim of this study was to evaluate the resistance of the membranes of PRF through different centrifugation protocols and intervals between application and use of membrane.

MATERIALS AND METHODS

This study was performed in line with the principles of the Declaration of Helsinki and approved by Human Research Ethics Committee under protocol number 3.552.132 (Uningá University, Maringá, Paraná, Brasil) and the patient signed an informed consent agreement.

To obtain the samples of this study were collected 300 mL of blood from a patient donator. The volunteer was a male with 34 years old, leukoderma and systemically healthy. In laboratory analysis, the sample obtained showed blood count and coagulogram values within the reference standard. The volume total was divided into 30 tubes, each one containing 10 mL.

The samples were divided into three groups, according to the g-force (F) applied during centrifugation:

Group 1: F200 m/s2

Group 2: F400 m/s2

Group 3: F800 m/s2.

Figure 1 shows the centrifuge acquired for this study, for centrifugations during 10 min of the all samples through centrifuge FibrinFuge® (Monteserrat, Changzhou Jiangsu, China). The physical characteristics of the membranes, independent of the applied force, were opaque yellow coloration in 2/3 from extension and red coloration in 1/3 from membrane. The membrane was 50 mm long and 2 mm thick, with a smooth texture and gelatinous consistency. Each group was subdivided into two situations: Immediate preparation of the membrane (T0) or preparation of the membrane with 30 min after centrifugation (T30). Independent of the waiting time, for membrane confection the blood concentrates were maintained into the metallic box for the time of 2 min and 30 s for pressing.

Figure 1

Centrifuge obtained to perform the processing of samples with different forces evaluated (FibrinFUGE 25, Montserrat®)

After making the membrane, immediately they were fixed by on a paper support [Figure 2]. An acrylic claw device [Figure 3] was developed to fixation of sample-support conjunct. The device was designed and cut in a universal testing machine with LASER SL1390 (Suda, Heifi, China). The sample-support conjunct was fixing into this device and coupled in a universal testing machine (Instron 5967, Canton, Massachusetts EUA) to perform the mechanical analysis.

Figure 2

Paper support developed to evaluate the membrane attached into longitudinal axis

Figure 3

Device developed to fix the sample-support on the universal testing machine

A 50N loading was applied to obtain g-force on the long vertical axis of the membrane. The traction force was applied until the total rupture of the membrane [Figure 4a-c]. After the disruption, the Bluehill 3 software (Canton, Massachusetts, USA) that was coupled in a universal testing machine system, registered the force necessary to initiate the rupture of the membrane.

Figure 4

(a) Membrane with traction; (b) Zoom of membrane with traction; (c) Membrane after rupture due the traction force applied

Scanning electron microscope images (JEOL JSM-7610F) were analyzed after rupture of membranes magnification × 50, ×1000 and ×2500 [Figures 5 and 6].

Figure 5

Leukocytes platelet-rich fibrin membrane after rupture with high magnification by Scanning Electron Microscope. (a) ×50; (b) ×1000; (c) ×2500 *Note the fibrin network

Figure 6

Leukocytes platelet-rich fibrin membrane after rupture (specifically the buffy coat area) with high magnification by Scanning Electron Microscope. (a) ×50; (b) ×1000; (c) ×2500

The data were collected and submitted for statistical analysis (IBM SPSS Statistic Software, Chicago, USA). To comparisons between resistance force (RF) of membranes obtained within different times were performed Student's t-test of independent sample; and to comparison of different forces, was performed ANOVA with post hoc Tukey’s, when applicable. The significance level of 5% was considered.

RESULTS

Table 1 shows the value of the RF required to break up the membrane considering the differences on applied force and waiting time evaluated. No significant difference was observed regarding to the RF of the membrane used in the different studied times (T0 and T30), for any force applied in the centrifugation, such as, F200, F400 and F800 (P = 0.2169, P = 0.8849 and P = 0.1172; Student's t test).

Table 1

The values of the resistance force groups (mean±standard deviation)

Groups	F200	F400	F800
T0	991.65±164.39	1.109.10±115.97	1.148.36±229.64
T30	996.39±225.06a	1.130.43±297.27a, b	1.313.91±64.97b

Different lower case letters on the same line indicate significant differences with significance level of 5% (student’s t-test). T0 – Immediate use (time); T30 – Use after 30 min (time); F200 – g-force of 200 m/s2; F400 – g-force of 400 m/s2; F800 – g-force of 800 m/s2

There was no significant difference in the membranes evaluated immediately after centrifugation, regardless of the force applied in the centrifugation process (P = 0.3750; Student's t-test).

On the other hand, after waiting time (30 min) for using the membrane, showed a significant increase in resistance (P = 0.040; Student's t-test) between F200 and F800 groups. Therefore, the membrane from F400 group showed no difference in resistance when compared to F200 (P = 0.762; Student's t test) and F800 (P = 0.317; student's t-test) with waiting time of 30 min.

DISCUSSION

The development of method of obtaining the blood concentrates by Choukroun et al.,[11] showed the evolution in search to increase the cell concentration and consequently the regenerative potential of this fibrin matrix. It was analyzed that when the centrifugation force is decreased, a blood concentrate is most porous and cellular and less dense.[912]

When the autologous blood is collected to membrane confection, no additives are incorporated into the material.[1314] The g-force is able to perform the segmentation of the blood layers, making it possible to obtain the membrane.[15] This procedure is relatively simple, can be performed at the clinic practice and has some advantages for bone grafts.[151617]

The use of PRF can be applied with different clinical fields, implant dentistry, surgery, and periodontology.[18] Otherwise, is known that the time and force performed in the centrifugation can influence the characteristics of the membrane that will be applied in the surgical receptor area.[819]

Membranes obtained from concentrates that are prepared with centrifugation force. It is calculated considering the size of the radius from centrifuge and the rotations per minute. A lesser centrifugation force shows greater release of growth factors and less degradation of fibrin.[19] In addition, lower centrifugation force results in a membrane with greater regeneration potential.[19202122] The biochemical characteristics influence the final composition of membrane to surgical approach or for another reason; the fact to increase membrane density is observed with centrifugation force[9] confirms to questions about the resistance of material. Therefore, this present was performed to aim to analyze the mechanical resistance of the membrane when subjected to traction (mN) on long axis according to both extremities until their rupture.

Although of preparation membrane process is considered a simple and easy procedure, some aspects should be considered.[1415] For surgical procedures, the membrane can be used during clinic practice, otherwise, the blood centrifuged can be prepared before begin the surgery or during the surgical procedure. In our study, we could observed that the waiting time did no showed influence regarding resistance membrane. Thus, the blood collect could be performed in the moment most convenient for the patient and professional.

On the other hand, when is stabilized that the waiting time to the preparation of membrane after centrifugation, it can be necessary to consider the force applied. In our results was observed that membranes obtained immediately after centrifugation showed no influence regarding resistance in function to applied force.

On the other hand, through the waiting time group analysis, the results showed a greater resistance of membrane when the centrifugation force was applied. It can suggest that when applied the waiting time with 30 min, the polymerization process begins with an acceleration format into membranes obtained with a major force, therefore, over time the influence of force in relation to resistance becomes remarkable. This membrane property with a major force of centrifugation shows higher density and less porosity[89] and it does not necessarily reflect in their resistance. Thus, for centrifugation with less force it can consider the presence of higher concentration of cellular components and growth factors,[9] whenever possible, immediate application provides to be a good viable alternative for procedures.

That protocol of centrifugation with less force will provide a greater concentration of membrane components without compromising the material resistance. However, more studies are necessary to prove this information and to evaluate other possible factors that may influence the resistance properties. Otherwise, clinical studies are essential to evaluate the impact of different resistances with waiting time for 30 min.

CONCLUSION

Within the limitation of this in vitro study, it can conclude that among the times evaluated, the collection and material centrifugation can be performed at the most appropriate time for the patient and professional. However, when there is a waiting time of 30 min after centrifugation, the applied force must be greater to increase the resistance of the membrane.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.