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. 2025 Oct 31;16(3):172–180. doi: 10.4103/ccd.ccd_348_25

Comparison of Fibrin Characteristics and Cellular Contents of Injectable Platelet-rich Fibrin Enriched Bone Block (“Sticky Bone”) Prepared Using Novel and Traditional Protocols – An In vitro Study

Sivanesan Karthikeyan Jagdish 1,✉, Antara Banerjee 2, Rajamony Eazhil 1, Amit Dey 2, Ganesh Kumar Anbazhagan 3, Asim Kanti Duttaroy 4, Ameer Khusro 5
PMCID: PMC12633828  PMID: 41281694

Abstract

Background:

Injectable platelet-rich fibrin (i-PRF) can be combined with bone graft materials to enhance its healing potential. Studies on improving the fibrin density and degradation properties of i-PRF-enriched bone grafts are lacking.

Aims and Objectives:

The aims and objectives of the study are to compare the fibrin porosity (density), solidification time, degradation properties, and cell counts of (i-PRF) bone blocks prepared using novel and traditional protocols.

Materials and Methods:

Venous blood (10 ml) was collected from six healthy volunteers to prepare the novel i-PRF in silica-coated plastic tubes (2000 g for 3 min) and traditional i-PRF in non-coated plain plastic tubes (700 g for 8 min). The i-PRF was mixed with xenograft to prepare the novel (NBB) or traditional bone blocks (TBB). The total solidification time and degradation properties of the bone blocks were analyzed. The density of the fibrin network in the bone blocks was compared using scanning electron microscopy images. The total and differential cell counts in the i-PRF were compared to those in whole blood.

Results:

The NBBs had higher fibrin density (10-fold) and solidified faster (3-fold) than the TBBs (P < 0.001). The NBBs were more stable and resistant to degradation (>10 days), whereas the TBBs degraded within 4–6 days (average 4.67 days). The cell counts of both platelets and leukocytes in the novel and traditional i-PRF were significantly higher than the whole blood control (P < 0.001).

Conclusion:

The novel protocol significantly enhanced the fibrin density, stability, and cell counts of the i-PRF bone blocks and shortened the solidification time.

Keywords: Bone graft, horizontal centrifugation, injectable-PRF, regeneration, sticky-bone

Introduction

Bone regeneration procedures in dentistry use autografts, xenografts, allografts, and alloplastic materials that aid in physiological bone remodeling.[1,2] The “PASS” principle highlights that primary wound closure, angiogenesis, graft stability, and space maintenance are crucial for successful bone regeneration. The loss of graft space and volume due to the disintegration of the graft material can lead to clinical failure.[3] The combination of platelet-rich fibrin (PRF) with bone grafts significantly improves the stability and regenerative potential of the graft material.[4,5,6] The PRF is an autologous second-generation platelet concentrate that is obtained without the use of anticoagulants.[7,8]

Solid PRF membranes are prepared using glass or silica-coated tubes.[9] In contrast, the injectable platelet-rich fibrin (i-PRF) is prepared using non-coated plain plastic tubes.[10,11,12] The platelets and leukocytes in the PRF are a rich source of autologous growth factors and cytokines that accelerate bone regeneration.[10,13,14] They also stimulate cell proliferation, migration, and differentiation, and promote soft-tissue healing.[15,16,17] Originally, fixed-angle centrifuges were used to prepare PRF.[9] Miron et al. demonstrated that utilizing horizontal centrifugation (swing-out rotors) in the preparation of PRF can lead to a significant increase in cell concentration and growth factor release.[18] The horizontal centrifugation distributed the cells uniformly throughout the PRF and minimized the trauma to the cells.[19,20] Miron et al. suggested that a horizontal centrifugation protocol of 700 g for 8 min resulted in the maximum cell distribution in the i-PRF.[20]

Few authors have used solid PRF membranes with bone grafts for bone regeneration procedures.[21] However, it has been shown that such preparations had low fibrin density with poor mechanical and degradation properties.[22] Liquid or i-PRF preparations are more frequently used with bone grafts than PRF membranes.[13,14,23,24] When mixed with the i-PRF, the graft particles are enmeshed in a fibrin network along with growth factors and cytokines to form a cohesive bone block. This bone block is also called “sticky bone” and was first described by Sohn et al. and Mourão et al.[13,14,23,24]

Currently, most studies are focused on improving the concentration of cells and growth factors in the i-PRF.[18,19,20,25,26] Studies on enhancing the fibrin density and degradation properties of i-PRF bone blocks are lacking. High-density fibrin is an ideal biomaterial with tissue-healing properties that can resist degradation of the bone grafts at the surgical site.[7,27,28,29] All the traditional protocols for preparing i-PRF bone blocks use only noncoated plain plastic tubes.[10,11,12,13,14,22,23,24] Many reports suggest that the clots formed using noncoated plain plastic tubes have low fibrin density and are not resistant to degradation.[22,30,31]

This study proposes a novel protocol that uses silica-coated tubes, high relative centrifugal force (RCF max-2000 g), and a short spin time (3 min) to prepare i-PRF bone blocks. Thus, the aims of the present study are (a) to compare the fibrin porosity (density) of the i-PRF bone blocks prepared using novel and traditional protocols, (b) to compare the solidification time and degradation properties of the novel and traditional i-PRF bone blocks, and (c) to evaluate the total and differential cell counts in the i-PRF prepared using novel and traditional protocols. The null hypothesis states that there is no difference in the fibrin porosity (density), solidification time, degradation properties, or cell counts between the novel and traditional protocols.

Materials and Methods

Study design and sample size estimation

This research was conducted as an in vitro study to compare the characteristics of i-PRF bone blocks prepared using novel and traditional protocols. All the procedures in this study involving human participants were performed following the ethical standards of the 1964 Declaration of Helsinki and institutional/national ethical guidelines. Ethical approval for the study was obtained from the Institutional Human Ethics Committee (Protocol Ref. No. IHEC-CDCRI/2024/FAC-0034).

A priori sample size estimation was performed using G*Power software (version 3.1.9.4). A two-tailed paired t-test (difference between two dependent means, matched pairs) was chosen with an alpha error probability of 0.05 and a desired power (1–β) of 0.90. The effect size (dz = 3.82) was calculated based on fibrin density data obtained from a previous study.[30] The analysis indicated that a minimum of 4 samples per group would be required. The sample size was increased to 6 per group to improve the robustness of the study.

Study participants

A total of 6 healthy volunteers (3 females, 3 males) visiting our outpatient dental clinic, aged between 20 and 40 years, were enrolled in this study after obtaining written informed consent. Before enrollment, the complete blood counts (CBCs) of the donors were assessed to ensure that their cell counts were within normal ranges. Volunteers with a history of smoking, alcohol consumption, bleeding disorders (genetic and acquired), abnormal platelet function, anemia, thrombocytopenia, and those under medications or treatment that interfere with blood coagulation and cell counts were excluded from the study.

Blood collection and centrifugation

The i-PRF was prepared using novel and traditional protocols. Two centrifugation devices of the same model (Medico Plus, Remi Laboratory Equipment, Mumbai, India) were used to prepare the samples simultaneously. Both these centrifuges had swing-out rotors with a radius of 110 mm (maximum RCF-2270 g) that could be used for horizontal centrifugation.[18]

Each of the six volunteers donated two blood collection tubes (10 ml) for each of the two protocols tested, resulting in a total of 4 tubes per participant. In addition, one tube (K2EDTA Vacuum Blood Collection tubes, Vacutech, Ref No: VKT210, Labtech Disposables, Ahmedabad, India) with anticoagulant (K2EDTA) was collected as a control for whole blood cell counts. Venous blood was collected from the antecubital vein with a 21-gauge butterfly needle (Scalp Vein Set, Romsons Scientific and Surgical Pvt., Agra, India) using a clinically approved method.[9] The first tube from each participant was used for preparing the i-PRF bone blocks to analyze solidification time, fibrin porosity (density), and degradation properties. The novel i-PRF was prepared using silica-coated plastic tubes (BD Vacutainer Serum Tube, Ref No. 367820, Becton Dickinson and Company, USA) and centrifuged at a maximum relative centrifugal force (RCF max) of 2000 g for 3 min. The traditional i-PRF was prepared using plain plastic vacutainer tubes (Vacutech i-PRF Vacuum Tube, Labtech Disposables, Ahmedabad, India) and centrifuged at 700 g (at RCF max) for 8 min.[20,22] The second tube was used for the analysis of cell counts. For this purpose, blood collection was done using plastic tubes for both protocols.[20] Centrifugation for each protocol was started within 1 min after blood collection to separate the blood into the plasma, buffy coat, and red blood cell (RBC) layers [Figure 1].

Figure 1.

Figure 1

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The separation of blood into layers following centrifugation using the novel protocol – 2000 g × 3 min (a) and traditional protocol – 700 g × 8 min (b)

Preparation of the injectable platelet-rich fibrin bone blocks

A xenograft (Osseograft, Advanced Biotech Products Pvt. Ltd, India) containing demineralized bone matrix (DMBM) was used for preparing bone blocks as follows:

  1. Novel bone block (NBB): The i-PRF from the silica-coated plastic tube was used to prepare the novel i-PRF bone blocks. The plasma was collected from top to bottom till the buffy coat layer using a sterile syringe, and 1 ml of the i-PRF was added drop by drop to 0.25 g of xenograft in a 35 mm petri dish. The graft was thoroughly mixed with the i-PRF for 15 s and shaped into a rectangular block

  2. Traditional bone block (TBB): The i-PRF from the plain plastic tube was used to prepare the traditional i-PRF bone blocks. The plasma was collected from top to bottom till the buffy coat layer using a sterile syringe, and 1 ml of the i-PRF was added drop by drop to 0.25 g of xenograft in a 35 mm petri dish. The graft was thoroughly mixed with the i-PRF for 15 s and shaped into a rectangular block [Figure 2].

Figure 2.

Figure 2

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A schematic to show the blood collection and analysis. The blood collected in a silica-coated tube (red cap) is centrifuged at 2000 g × 3 minutes to prepare the novel Injectable platelet-rich fibrin (i-PRF) (a). The blood collected in a plain plastic tube (yellow cap) is centrifuged at 700 g × 8 minutes to prepare the traditional i-PRF (b). The i-PRF prepared is then mixed with 0.25 g of bone graft to form the respective bone blocks. I-PRF: Injectable platelet-rich fibrin

Solidification time of injectable platelet-rich fibrin bone blocks

The solidification time of the bone blocks was measured using the method described by Feng et al.[22] The 35 mm petri dishes were gently tilted at 45° every 15 s to observe if the graft particles mixed with the i-PRF would separate from the bone block. The material was deemed to be solidified as a bone block if the graft particles did not separate from the cohesive mass. The total time taken for the complete solidification of the bone blocks was recorded in minutes.

Scanning electron microscopy and measuring fibrin porosity (density)

After solidification, the excess uncoagulated serum was removed from the 35 mm petri dishes by gentle blotting using a sterile gauze. The bone blocks were fixed using 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer for 24 h.[22] The samples were then rinsed with 0.2M sodium cacodylate buffer and distilled water. The samples were dehydrated using serial dilutions of ethanol (25%, 50%, 75%, 95%, and 100%) and then dried at 37°C in an oven.[22] The dried samples were sputter-coated (SC7620 mini Sputter Coater, Quorum Technologies Ltd, UK) with gold–palladium. Two regions from each bone block were captured using scanning electron microscopy (SEM) (Sigma 300 SEM, Carl Zeiss, Germany) at ×10000 magnification.

The SEM images (8-bit grayscale) made at ×10000 magnification were analyzed using the open-source image analysis software (ImageJ, U.S. National Institutes of Health, Bethesda, Maryland, USA) to calculate the porosity of the fibrin network.[32] The images were first set to scale using the scale bar. The SEM images were cropped to the region of interest to exclude the scale bar before segmentation and then binarized using the manual thresholding function. The segmentation that best represented the original image was chosen to calculate the mean pore area and the percentage porosity in the fibrin network. The area that was not segmented as a fiber (background) represented the pore space and was expressed as percent porosity.[32,33] Fibrin density refers to the extent to which the fibers are tightly packed together. Consequently, samples exhibiting lower porosity were deemed to possess greater density.[33] A representation of the segmentation of SEM images is shown in Figure 3. All the images were analyzed by two independent evaluators, and the average of the two measurements was taken for analysis. The inter-rater reliability was assessed using the intraclass correlation coefficient (ICC). The ICC estimate and the 95% confidence interval were calculated using SPSS Statistical Software (IBM corp. Statistical Package for Social Sciences Software for Windows; Version 30.0, Armonk, NY, USA), applying a two-way random effects model with average measurements and a consistency definition.

Figure 3.

Figure 3

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A sample workflow for segmentation of scanning electron microscopy images using Image J software is presented in the figure. The original 8-bit uncropped image (a) is cropped to the region of interest (b), The manual thresholding function is then applied for image segmentation (c) into fibers and background (red). The area that was not segmented as a fiber (red) represented the pore space and was expressed as percent porosity

Degradation of the injectable platelet-rich fibrin bone blocks

The bone blocks were covered with 3 ml of phosphate-buffered saline solution supplemented with 0.05% trypsin-EDTA. Trypsin is a protease that can degrade fibrin in vitro and hence was used in our study.[34] The samples were incubated at 37°C in a humidified atmosphere and 5% CO2 for 7 days. Photographs of the samples were taken every 24 h to evaluate the disintegration of the bone blocks.[22]

Automated cell counting

For cell counts, the liquid plasma was harvested using a pipette from top downward to include the buffy coat along with 0.5 ml of the RBC layer. The harvested liquid fraction was then transferred to a tube with an anticoagulant (K2 EDTA). The anticoagulation was performed only for research purposes to enable cell counting.[20] The i-PRF samples and the whole blood control were sent for CBCs using an automated hematology analyzer (DxH 900, Beckman Coulter, California, USA) based on the Coulter Principle. The total counts of platelets, leukocytes, RBCs, neutrophils, lymphocytes, and monocytes were recorded. The data were summarized graphically using Graphpad Prism software (Version 10.4.1).

Statistical analysis

The data were tested for normality using the Kolmogorov–Smirnov test with the Dallal-Wilkinson-Lilliefor P value. Statistical analysis was done using GraphPad Prism software (Version 10.4.1). All data were expressed as mean and standard deviation. The data for solidification time and porosity were analyzed using the Student’s-t-test. The data for cell counts were analyzed using One-way analysis of variance, followed by Tukey’s post hoc test for multiple pairwise comparisons. Non-normal data for cell counts were analyzed using Kruskal–Wallis’s test, followed by Dunn’s multiple comparisons test. Differences between the groups were considered statistically significant if P < 0.05 (*P < 0.05), 0.01 (**P < 0.01), or 0.001 (***P < 0.001).

Results

The data for solidification time, fibrin density, and cell counts of platelets, leukocytes, lymphocytes, neutrophils, and monocytes were found to be normally distributed (P > 0.1000). The RBC counts were not normally distributed (P < 0.1000).

Solidification time

The solidification times (in minutes) for the NBB and TBB are shown in Table 1. The difference in solidification times between the two bone blocks was statistically significant (P < 0.001). The solidification time of NBB’s (6.25 ± 0.89 min) was 3-fold faster than TBB’s (21.17 ± 2.70 min).

Table 1.

Comparison of solidification time of injectable platelet-rich fibrin bone blocks in minutes

Samples (n) Mean±SD Mean difference 95% CI of mean difference t Degrees of freedom (df) P
TBB 6 21.17±2.70 14.92 11.70–18.13 11.94 5 0.00007***
NBB 6 6.25±0.89
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***Statistically significant difference. Data were analyzed using paired Student’s t-test. i-PRF: Injectable platelet-rich fibrin, NBB: Novel i-PRF bone block; TBB: Traditional i-PRF bone block; CI: Confidence interval. SD: Standard deviation

Scanning electron microscopy and fibrin porosity (density)

The SEM images showed an interconnected network of fibrin around the graft particles. The dense fibrin network formed a mesh binding the particles to form a bone block in both groups. At ×10000 magnification, the fibrin network around the NBB was found to be denser with less porosity compared to the TBB [Figure 4]. The total area of the porosity in the NBB and TBB was 2.3% ± 0.71% and 20.23% ± 9.88%, respectively. The difference was found to be statistically significant (P < 0.01) with the NBB having a 10-fold lesser porosity than the TBB [Table 2]. The ICC estimate was found to be 0.995, indicating excellent reliability of the measurements [Table 3]. These findings suggest that the fibrin density in the NBB was significantly higher than in the TBB.

Figure 4.

Figure 4

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Representative scanning electron microscopy images of the bone blocks showing the fibrin architecture at a magnification of × 10000 are shown in this figure - (a) fibrin mesh in the novel bone block, (b) fibrin mesh in the traditional bone block

Table 2.

Comparison of fibrin density (porosity) as area percentage in the injectable platelet-rich fibrin bone blocks

Samples (n) Mean±SD Mean difference 95% CI of mean difference t Degrees of freedom (df) P
TBB 6 20.33±9.88 18 7.68–28.31 4.48 5 0.0065**
NBB 6 2.33±0.71
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**Statistically significant difference. Data were analyzed using paired Student’s t-test. i-PRF: Injectable platelet-rich fibrin, NBB: Novel i-PRF bone block; TBB: Traditional i-PRF bone block; CI: Confidence interval. SD: Standard deviation

Table 3.

Inter-rater reliability of porosity measurements using the intraclass correlation coefficient

ICC type Estimate 95% CI
 F test with true value 0
Lower bound Upper bound Value df1 df2 P
Average measures 0.995 0.982 0.999 196.369 11 11 <0.001
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ICC was calculated in SPSS software (version 30) using a two-way random effects model, consistency definition, and average measures. CI: Confidence interval; ICC: Intraclass correlation coefficient; SPSS: Statistical Package For The Social Sciences

Degradation of bone blocks

The degradation of the i-PRF bone blocks placed in PBS with trypsin-EDTA over time is shown in Figure 5, and the data are summarized in Table 4. The TBBs were completely degraded and lost their initial shape within 4–6 days (average of 4.67 days). In the NBBs, a few graft particles from the periphery were found to separate from the bone blocks. However, they did not lose their shape significantly. The NBBs were stable and retained their shape over 10 days.

Figure 5.

Figure 5

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Representation of the degradation of the injectable platelet-rich fibrin bone blocks placed in phosphate buffer saline with 0.05% trypsin-EDTA over time. NBB: Novel i-PRF bone block; TBB: Traditional i-PRF bone block

Table 4.

Degradation of the injectable platelet-rich fibrin bone blocks

Samples (n) Average degradation time (days)
NBB TBB
6 >10 4.67
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NBB: Novel i-PRF bone block; TBB: Traditional i-PRF bone block

Cell counts

The cell counts in the novel and traditional i-PRF compared to whole blood (control) are shown in Figure 6. Both novel and traditional protocols led to a significantly greater accumulation of platelets and leukocytes compared to the control (P < 0.001). The traditional and novel protocols resulted in an over 2-fold and 1.5-fold increase in the platelets, respectively, compared to the control (P < 0.001). The platelet count in the traditional protocol was found to be higher than in the novel protocol (P < 0.001). The RBC counts were significantly lower in both the traditional protocol (P < 0.05) and the novel protocols (P < 0.01) compared to the control. No significant differences were found in the leukocyte, RBC, neutrophil, lymphocyte, and monocyte count between the two protocols (P > 0.05).

Figure 6.

Figure 6

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Distribution of various blood cells in novel versus traditional injectable platelet-rich fibrin compared to whole blood control. I-PRF: Injectable platelet-rich fibrin. Statistically significant differences - *P < 0.05, **P < 0.01, ***P < 0.001. ns: Not statistically significant (P>0.05)

Discussion

The present study investigated the characteristics of novel i-PRF bone blocks prepared using silica-coated plastic tubes, high RCF (2000 g), horizontal centrifugation, and a short spin time (3 min). The findings of our study demonstrated significant differences in the fibrin density, solidification time, degradation properties, and platelet counts between novel and traditional protocols. However, there were no significant differences in the other cell counts. Hence, the null hypothesis was only partly accepted.

The traditional protocols use plain noncoated plastic tubes with hydrophobic surfaces to prepare i-PRF bone blocks.[10,11,12,13,14,22,23,24] The use of plain plastic tubes delays the coagulation of the i-PRF due to the absence of clot activators.[31,35] The fibrinogen in the traditional i-PRF remains unpolymerized and is maintained in the liquid state until the activation of the coagulation cascade. When this i-PRF is combined with a bone graft material, the collagen in the DMBM triggers the intrinsic coagulation pathway, facilitating the conversion of fibrinogen into fibrin.[35,36] In the novel protocol, clot activators such as silica or glass trigger the coagulation cascade through the intrinsic pathway.[21,31] In the novel protocol, the polymerization of fibrinogen to fibrin is initiated by the silica or glass coating of the tubes rather than the collagen in the bone graft.

Recently, the use of silica-coated tubes for the preparation of PRF has been a subject of controversy. Silica-coated tubes have been used for a long time for PRF preparations due to the unavailability of glass tubes from commercial manufacturers.[7,8,30] Few in vitro studies have raised concerns regarding the contamination of PRF preparations due to silica and its potential acute cytotoxic effects.[37,38,39] In contrast, other in vitro studies have reported stimulation, proliferation, and differentiation of a wide range of human cells exposed to PRF prepared in silica-coated tubes.[15,16,17] Silica has been shown to have toxic effects when inhaled.[40] Nevertheless, acute in vitro cytotoxic effects are seen only at extremely high concentrations.[16,41] Furthermore, there is a lack of in vivo studies, case reports, or clinical trials documenting acute or chronic tissue reactions associated with PRF prepared using silica-coated tubes. A systematic review of clinical studies concluded that postoperative complications such as pain, swelling, soft-tissue healing, and alveolar osteitis after grafting with PRF, were not affected by the type of tube material (silica or glass).[42] Hence, silica-coated tubes continue to be used for PRF preparations until concrete evidence contradicts their use.

The solidification time of the TBB reported in our study (21.17 ± 2.70 min) is concurrent with previous literature.[13,22,23,24] Feng et al. found that the solidification time can be reduced to 2 min by incorporating a solid PRF membrane along with i-PRF.[22] This requires two PRF preparations (solid and liquid) and two test tubes (glass and plain tubes) with a centrifugation time of 8 min for each preparation. Using the novel protocol, the total solidification time was only 6.25 ± 0.89 min. The novel protocol provides adequate time for clinical handling of the graft material while simultaneously decreasing the total duration of the surgical procedure. Using the novel protocol, two tubes (glass and plain tubes) are unnecessary for preparing i-PRF bone blocks.

Platelet concentrates with a high-density fibrin network are essential for cell migration and cytokine enmeshment.[7,28,29] The fibrin density is dependent on the fibrinogen concentration in the platelet concentrate.[7,27] The findings of the study by Kargarpour et al. suggest that the main source of “clottable” fibrinogen in i-PRF bone blocks or “sticky bone” was from the upper plasma layers, while the buffy coat was more of a cell source.[35] The novel protocol aims to collect the upper plasma layers, the buffy coat layer, and 0.5 ml of the RBC layers to harvest maximum cells and liquid fibrinogen. However, these layers must be harvested when the fibrin is still in the unpolymerized liquid form. To achieve this, a short spin time of 3 min was proposed. The rationale for using a high RCF (2000 g) and horizontal centrifugation was to achieve greater cell separation and maximum cell counts within this short spin time.[18,25]

The findings of our study indicate that the novel protocol significantly decreased the porosity and increased the density of the fibrin network in the bone blocks by 10-fold compared to the traditional protocol. We also noted a corresponding increase in the stability of the NBBs (>10 days) compared to the TBBs (average 4.67 days). The greater stability of the NBBs could have been due to the low porosity and higher fibrin density in the NBBs compared to TBBs.[22] As far as the authors are aware, there is a lack of studies that have measured the porosity or density of fibrin in i-PRF bone blocks using SEM images. Nonetheless, the study by Feng et al. revealed distinct morphological variations in the SEM images of various types of i-PRF bone blocks made using solid (700 g, 8 min, glass tubes) and liquid (700 g, 8 min, plain plastic tubes) horizontal centrifugation protocols.[22] Their results showed that bone blocks made using liquid PRF alone had lower fibrin density than those made with a mixture of solid and liquid PRF. Furthermore, it was observed that bone blocks made using liquid PRF alone disintegrated within 96 h, unlike those produced with both solid and liquid PRF.[22] Results from our study are consistent with these findings. In another study, Silva et al. reported that PRF clots made using plain plastic tubes had significantly lower fibrin density than those obtained using glass or silica-coated tubes.[30] This provides additional evidence that the fibrin density is significantly higher in clots formed with silica-coated tubes compared to those prepared with plain plastic tubes.

The accumulation of platelets and leukocytes in the i-PRF is influenced by variations in the centrifugation speed, time, and the method of harvesting the i-PRF.[18,20,26] We observed a significantly greater accumulation of platelets in both the novel and traditional i-PRF compared to the whole blood. Our results are consistent with those reported in prior studies despite variations in the centrifugation protocols.[11,18,20,26,43] Miron et al. reported that using horizontal centrifugation, a protocol of 700 g for 8 min presented the maximum yield of platelets and leukocytes distributed evenly throughout the liquid PRF layers.[20] In the present study, platelet counts were higher with the traditional protocol compared to the novel protocol. Previous reports have demonstrated that longer centrifugation times enhance cell separation and increase platelet yield.[18,20] Therefore, the relatively lower platelet counts observed with the novel protocol may be attributable to its shorter centrifugation time.[20] Furthermore, Silva et al. reported that i-PRF prepared in plain plastic tubes exhibited higher platelet counts, likely due to the hydrophobic properties of the tube material.[30] These factors may have contributed to the reduced platelet counts observed with the novel protocol. Nevertheless, this remains speculative and requires further investigation.

In contrast to platelet counts, previous studies have reported variations in the leukocyte counts associated with traditional i-PRF. Most of them have reported an increase in leukocyte counts.[11,18,20,26] The study by Serafini et al. found a significant decrease using the traditional i-PRF.[43] This could be because of differences in the harvesting of i-PRF layers.[43] Miron et al. demonstrated that the maximum concentration of leukocytes was within the 0.3–0.5 ml of buffy coat near the RBC layer, primarily due to their higher densities compared to the platelets.[20,26] Hence, omitting this layer could lead to low leukocyte counts in i-PRF. In our study, we collected the i-PRF, including 0.5 ml of the RBC layers, to harvest the maximum concentration of leukocytes. We found no difference in the leukocyte or RBC counts between the two protocols.

The novel protocol is simple without the need for any additional equipment, plain non-coated tubes, or multiple centrifugation protocols for preparing the i-PRF bone block. The high-density fibrin network with lesser porosity improves the stability and decreases the degradation of the i-PRF bone block. The i-PRF bone block is also moldable and can be shaped to the desired form in the defect area. The novel protocol effectively meets all criteria of the “PASS” principle by achieving a high concentration of platelets and leukocytes, alongside improved graft stability and reduced disintegration.

This study has several limitations. The sample size was relatively small, and the study design was in vitro, necessitating in vivo confirmation through further clinical studies. The enzymatic degradation challenge was also performed only in vitro and may not fully reflect in vivo conditions, which warrants additional research. Faster clotting time may also limit the technique, as blood collection and centrifugation must be carried out rapidly, within 60–90 s. The i-PRF obtained cannot be stored for long periods and must be mixed with the graft immediately. Moreover, the influence of silica concentration in collection tubes, which may affect coagulation, was not evaluated in this study and should be explored in future investigations. Overall, further clinical studies are required to substantiate the graft stability and the potential for clinical bone regeneration with this technique.

Conclusion

The novel protocol significantly enhanced the fibrin density, stability, and shortened the solidification time of the i-PRF bone blocks. The novel i-PRF protocol was also able to achieve a high concentration of both platelets and leukocytes.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.

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