Structure,Barrier Function,and Bioactivity of Platelet-Rich Fibrin Following Thermal Processing

Abstract

Platelet-rich fibrin (PRF) has been utilized as a substitute for resorbable membranes during guided bone regeneration therapy as it is a more bioactive biomaterial with living cells and growth factors than resorbable membranes. Nevertheless, PRF poses obvious disadvantages in its mechanical strength since its rapid degradability has been shown to typically resorb within a 2-week time period. In the present study, the barrier function and biological and mechanical properties of PRF were investigated both as standard therapy and after thermal processing. Two heating processes were applied: both single-side heating and double-side heating at 90°C for 10 s using a metal plate heater. The appearance and weight of PRF membranes were documented after heating, along with their morphological and mechanical properties evaluated by scanning electron microscope and tensile strength tests. The viability of cells found within PRF membranes was also evaluated using live/dead cell viability and CCK-8 (cell counting kit-8) assays. To comprehensively evaluate the barrier function of PRF membranes, Hoechst staining of human gingival fibroblasts, which can be distinguished from cells within the PRF membrane by emitting blue light at an excitation wavelength of 488 nm, was seeded onto the surface of PRF membranes. Furthermore, osteoblasts were cultured with extracts from different PRF groups to evaluate the biocompatibility of PRF membranes. The degradation rate of PRF membranes was examined by digestion assay. Compared with the nonheated PRF control, the size and weight of PRF membranes led to a significant decrease with a denser PRF microstructure following heating. In summary, the double-sided heating of PRF membranes not only demonstrated an improvement in mechanical and degradation properties but also led to a decrease in cell viability and proliferation.

Impact statement

Platelet-rich fibrin (PRF) is getting more and more attention in the field of guided bone regeneration, especially used as the barrier membrane. However, the rapid degradation property restricts its usage. It has been found that heating, as a nonadditive treatment method, can prolong the degradation time of PRF in vivo and in vitro, but most of the current research on PRF heating treatment is limited to research about the degradation time of PRF membrane. Our work herein systematically and comprehensively evaluated the structure, mechanical properties, barrier function, and bioactivity of the PRF membrane after heating treatment.

Introduction

Dental implants have become a vital option for patients with tooth loss, with resulting alveolar bone deficiencies often posing a challenge before implant placement.¹ Guided bone regeneration (GBR), which was introduced in 1976 by Melcher, is one of the most common solutions for bone augmentation at the implant site.² During GBR procedures, barrier membranes play a pivotal role by preventing the invasion of faster-growing soft tissue cells into the slower-growing bony tissue area with a defect.^3–5 An ideal GBR membrane should have the following characteristics: (i) mechanical stabilization, (ii) biocompatibility, (iii) occlusive function, (iv) appropriate resorption time after hard tissue healing, and (v) easy handling.^6,7

At present, two types of barrier membranes are available for clinical practice: nonresorbable and resorbable membranes.⁶ While originally nonresorbable membranes were more frequently utilized, they require a second surgical procedure for removal, causing additional patient morbidity; also, certain types are more likely to cause membrane exposure due to their stiffness. For these reasons, absorbable membranes were developed.^8,9 However, resorbable membranes have a disadvantage in that, generally speaking, they have poorer mechanical properties and greater variability in their resorption rates. Regardless of the membrane type, current barrier membranes act as passive barriers because they have limited regenerative potential without active cells or bioactive growth factors to help further speed tissue regeneration.¹⁰

Platelet-rich fibrin (PRF), which was initially developed in 2001, has been widely used in dental practice for a variety of procedures, including sinus lifting, ridge preservation, and periodontal regeneration.^11–16 PRF is composed of three major components that are autologous and beneficial for tissue regeneration: live cells, including platelets and leukocytes; growth factors, including vascular endothelial growth factor, transforming growth factor, and platelet-derived growth factor; and a three-dimensional fibrin structure for binding growth factors and supporting cell in-growth.¹⁷ Recently, solid PRF clots have been extracted followed by subsequent flattening and utilized in guided tissue regeneration/GBR procedures.^18–21

PRF membranes provide many advantages over other resorbable barrier membranes as they are completely autologous and further release regenerative growth factors to promote tissue healing.^22,23 PRF membranes are prepared by a simple venous blood draw in glass tubes without anticoagulant and therefore do not elicit any foreign body reaction (as some commercial barrier membranes do).^24–26 Moreover, recent studies have demonstrated that the incorporation of living host-immune leukocytes within PRF membranes can inhibit pathogenic bacteria such as Staphylococcus aureus and Escherichia coli found in the oral cavity,^27–29 which can be beneficial in minimizing postoperative infections following GBR procedures.

One of the drawbacks of PRF when compared to other resorbable membranes, such as collagen membranes, is its rather short resorption period ranging from 1 to 2 weeks. The rapid degradability and poor mechanical properties restrict their application in standard GBR procedures.^30,31

Recently, it was revealed that the resorption properties of PRF could be drastically extended by heat treatment of the PRF membrane.^32–34 In original studies on this topic, the heat-compression technique delayed the degradation of PRF membranes with user-friendly preparation processes available from clinical applications.^32,34 Typical membranes are prepared by heating albumin gel with liquid-PRF by first heating the upper liquid platelet-poor plasma layer and mixing it back with the liquid buffy coat zone (liquid PRF).^33–35

Therefore, the aim of the present study was to evaluate the intrinsic properties of PRF following various heat treatment modalities (one side of the membranes vs. two-sided), including its mechanical properties, fibrin networks, cell viability, barrier function, and degradation ability.

Materials and Methods

Blood collection, PRF membrane, and PRF extract preparation

All the procedures carried out in this study with human participants were approved by the Ethics Committee of the School and Hospital of Stomatology, Wuhan University (B52/2020). Peripheral blood samples were collected from eight healthy volunteers aged 18–30 years with no anticoagulant or antibiotic usage for at least 3 months before blood collection without using anticoagulants. Each of the eight volunteers donated four blood collection tubes (10-mL glass tubes) each time. The horizontal centrifugation protocol (10 mL, 700 g, 8 min) according to previous studies was performed using a horizontal centrifuge (Eppendorf 5702 centrifuge, Germany).³⁶

For preparation of the PRF extracts, each treated PRF membrane was transferred to a six-well plate and incubated with 5 mL Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 1% antibiotics (Gibco, Thermo Fisher Scientific) in a 37°C incubator for 3 days. Then, the medium was filtered and stored at 4°C as PRF conditioned media/extracts for future experiments.

Heating treatment of the compressed PRF membrane

After gently removing the red blood cells from standard PRF clots, they were compressed about 1 min into standardized membranes that were ∼1 mm thick using metal equipment kits. All instruments used in the heating process were sterilized by ultraviolet rays. The PRF membranes in the control groups were not heated. The PRF membranes in the test group were placed on a metal plate heater (H₂O₃-H; Coyote Bioscience Yixing Co., Ltd.), which has a dry metal surface that can be adjusted to 90°C, heated for 10 s (either one sided or two sided) as previously described.³²

PRF size and quality measurement

Each PRF membrane was measured with a Vernier caliper and weighed. ImageJ software (version 1.8.0; Fiji) was used for the calculation of membranes' area. The weight of membranes was calculated using analytical laboratory balances (ME204; Mettler Toledo). The appearance of the PRF membranes is shown in Figure 1A. Each group was repeated with three replicates.

FIG. 1.

The appearance and change in solid-PRF membranes after various heat-treatments. (A) The metal plate heater was utilized. (B) Images of PRF membranes treated by different heating forms. (C) Changes in PRF membrane area after heat treatment. (D) Changes of PRF membrane weight following heat treatment. Statistical differences were compared between the control nonheated PRF group and the other heating groups. ns: not statistically significant versus control group; *p < 0.05 and ***p < 0.001. PRF, platelet-rich fibrin.

Scanning electron microscope

The samples were fixed with 2.5% glutaraldehyde (Merck, Darmstadt, Germany) at room temperature for 4 h and then dehydrated with gradient ethanol (25%, 50%, 75%, and 100%) for 30 min at each concentration. Afterward, critical point drying and gold sputter coating were performed. The morphology of surfaces and cross sections of each sample was observed using a field emission scanning electron microscope (Zeiss; Sigma) under a 20 kV acceleration voltage of the electrons and a working distance of 7 mm. The magnification ranged from 2000 × to 6000 × at 6 different field views for each sample.

The inner cell viability of PRF membrane

Each treated PRF membrane was transferred into a six-well plate, washed with phosphate-buffered saline (PBS), and then stained using a calcein-AM/PI double stain kit (40747ES80; Yeasen). Then, 15 μL Calcein-AM solution and 45 μL PI solution were added to 5 mL 1 × assay buffer to prepare the assay solution. Two milliliters of the assay solution was then added to each well and incubated at 37°C for 30 min. Samples were washed with PBS three times for 5 min. Images of the surfaces and cross sections of each of the PRF membrane groups were captured using a fluorescence microscope (Oxion Fluorescence, The Netherlands) with excitation at 488 nm and detection at 530 and 620 nm. Calculate the number of dead and living cells in five pictures on each surface of each group.

Thereafter, cells were expressed as percentages of live versus dead cells. Cell Counting Kit-8 (CCK-8; Dijindo) was also utilized to evaluate the cell viability in PRF membranes. In brief, 0.1 g PRF samples were fractionated into small pieces with sterile surgical scissors and then cultured in 1 mL DMEM with 10% FCS and 1% antibiotics (Gibco, Thermo Fisher Scientific). At 3 days, 100 μL of CCK-8 solution was added to each well and incubated for 30 min at 37°C in the dark. The medium was then pipetted and measured at 450 nm using a microplate reader (PowerWave XS2; BioTek, Winooski) as previously described.³⁴

Mechanical strength test

The mechanical properties of the PRF membranes were measured using a tension meter (ZQ-990; ZhiQu) at a speed of 1 mm/min under standard ambient conditions at 25°C ± 3°C room temperature and 50% ± 25% relative humidity. Before stretching, the initial length between the two clamps was set to 5 mm. The strain-stress curve, the maximum tensile strength, and the tensile strain at break were recorded. Each group was repeated with three replicates. The maximum stress was calculated by dividing the force by the initial tissue cross-sectional area. The tensile strain at break was defined as tensile strain at the tensile stress at break. The strain-stress curve was considered as the modulus of elasticity in tension.

Cell culture

Human gingival fibroblasts (HGFs) were derived from normal gingival tissue from four human donors undergoing third molar extraction after signed ethical approval and informed consent as previously described.³⁷ Gingival samples were rinsed three times with PBS (150 mM NaCl, 20 mM sodium phosphate, pH 7.2) that contained 100 U/mL penicillin G (Thermo Fisher Scientific, Inc.) and fractionated into small pieces with sterile surgical scissors. Pieces of human gingival tissue were then subsequently transferred into T25 culture flasks with α-MEM in an incubator at 37°C containing 5% CO₂ for 2 h. After the cells were able to adhere, 3 mL of α-MEM containing 20% fetal bovine serum (FBS; Gibco, Life Technologies Corporation) with 1% antibiotics was added and cultured in an incubator at 37°C under 5% CO_2.

After 7 days, cell confluency was reached, and HGFs were trypsinized and further passaged in α-MEM with 10% FBS. Human fetal osteoblasts (hFOB) were purchased (LSMCE022) and cultured in DMEM with 10% FCS and 1% antibiotics (Gibco, Thermo Fisher Scientific). All cell culture medium was changed every 2 days throughout the cell culture experiments. Use Invitrogen Countess 3 (Thermo Fisher Scientific, Inc.) to count the cells of the cell suspension, then apply it to subsequent related experiments after dilution.

Barrier function test

Nuclear staining of HGFs was performed using Hoechst (20 mM; Thermo Fisher Scientific, Inc.) for 30 min at 37°C in the dark. Stained HGFs were then rinsed three times with PBS (150 mM NaCl, 20 mM sodium phosphate, pH 7.2). PRF samples were placed on adhered glass slides in 6-well plates, and then 200 μL of pretreated HGF suspension was added directly to the PRF samples at a density of 10,000 cells per membrane and cultured for 2 h to allow adherence. Then, 3 mL DMEM containing 10% FBS was gently added. Images of the surfaces of the PRF membranes and slides were then captured using a fluorescence microscope (Oxion Fluorescence) with an emission set at 488 nm. Thereafter, number of nuclear staining cells were calculated in five pictures on each surface of each group.

Cell proliferation assay

To avoid the insignificant differences between the groups due to excessive cell proliferation in the later stage, human osteoblasts (LSMCE022) suspension were counted by Invitrogen Countess 3 (Thermo Fisher Scientific, Inc.) and then were diluted to 1000 cells per milliliter and then seeded 100 μL/well in 96-well plates as previous study recommended.³⁷ The culture medium using in this test were DMEM with 10% FCS and 1% antibiotics (Gibco, Thermo Fisher Scientific) with or without 20% conditioned media from the PRF membranes. Five sets of duplicate wells were used in each group to minimize the error due to the initial cell volume. At 1, 3, and 5 days, 10 μL of CCK-8 solution was added to each well and incubated for 30 min at 37°C in the dark. The medium was then pipetted and quantified using a luminescence plate reader (TECAN Infinite 200 Pro) at 450 nm.

In vitro degradation test

PRF membranes were freshly prepared and inserted in 12-well plates. Before adding culture medium, all the PRF membranes were repeatedly rinsed with PBS (150 mM NaCl, 20 mM sodium phosphate, pH 7.2) to eliminate serum. Then the samples were incubated in a CO₂ incubator with DMEM supplemented with 20% human plasmin (Hematologic Technologies, Essex Junction, VM). The initial weight of each PRF membrane is about 0.05 g. After taking out the PRF membrane from the 12-well plates, used a sterilize gauze to absorb the excluded and then calculated it using analytical laboratory balances (ME204; Mettler Toledo). The membranes were put back into the original medium immediately after weighing. The appearance and weight were photographed and examined and recorded once per day.

Statistical analyses

Each experiment was performed with at least three replicates in all experiments. GraphPad Prism software 8.0.2 was utilized to analyze the data. To test the significance of the observed differences between groups, the data were analyzed using one-way analysis of variance (ANOVA) for the inner cell viability of PRF membrane evaluation, mechanical strength test, barrier function test, and cell proliferation assay. Two-way ANOVA was utilized for the PRF size and quality measurements and the in vitro degradation test. *p < 0.05, **p < 0.01, ***p < 0.001 was considered statistically significant.

Results

Macroscopic and microscopic observations of PRF

After heat treatment, the macroscopic surface of PRF membranes changed from a more translucent to opaque color and texture (Fig. 1A). Heating also reduced the area and weight of PRF membranes (Fig. 1B, C). The microstructure observation of each PRF membrane performed by scanning electron microscopy (SEM) demonstrated that the fibrin in the control group and the nonheated surface of the single-side heating group showed a network with fibrin bundles (Fig. 2). However, when heat treatment was applied to the PRF membranes, the structure of the fibrin was much more condensed (Fig. 2). The cross sectional analysis by SEM also revealed that the heating treatment densified the fibrin structure and reduced the number of visible cells within the PRF membrane.

FIG. 2.

SEM images of PRF membranes after various heat treatments. (A, E) Control PRF membranes. (B, F) Nonheated surface of the single-side heat-treatment PRF group. (C, G) Heated surface of single-side heat-treatment PRF group. (D, H) Double-sided heat treatment PRF group. (I) The cross sections of control group, (J) single-side heat-treatment PRF group and (K) double-sided heat treatment PRF group. The surface of each PRF membrane was scanned using 2k × magnifications. The cross sections of the samples were monitored using 6k × magnification. SEM, scanning electron microscopy.

Internal cell viability of PRF membranes after heat treatment

We then evaluated the viability of cells within the PRF membranes under various heat treatment modalities by using live/dead and CCK-8 assays. The fluorescence microscopy images demonstrated that cells in the double-sided heat-treatment group were more similar to that at the periphery (near the heated surfaces) (Fig. 3A–I). The single-sided heat treatment group retained many more living cells, especially toward the nontreatment side. The proliferation assay performed using the CCK-8 assay also demonstrated significantly more living cells in the single-sided heat treatment group than in the double-sided heat treatment group (Fig. 3J).

FIG. 3.

Fluorescence microscope images following a live/dead assay. The merged fluorescent images of the live/dead staining with viable cells appearing in green and dead cells in red. (A, E, K) Control PRF membranes. (B, F, L) Nonheated surface of the single-side heat-treatment PRF group. (C, G, N) Heated surface of single-side heat-treatment PRF group. (D, H, M) Double-sided heat treatment PRF group (magnification, 10 × ). (I) Average ratio of live cells versus dead cells in the various PRF groups after heat treatment. (N = 5). (J) Results of a CCK-8 assay to evaluate the survival rate of cells in the PRF groups after various heat treatments. Statistical differences were compared between the control nonheated PRF group and the other heating groups. ***p < 0.001. CCK-8, Cell Counting Kit-8. Color images are available online.

Mechanical strength of PRF membranes after heat treatment

The mechanical strength of PRF membranes was characterized by maximum stress, tensile strain at break and stress-strain curves. In the single-sided heat treatment group and double-sided heat treatment group, the maximum stress of the PRF membranes was significantly improved when compared to standard nonheated controls (Fig. 4A). No significant differences were found in strain at break between the single-sided and double-sided heat treatment groups; however, both were significantly superior to the nonheated control group (Fig. 4B). The stress-strain curve showed that PRF membranes in the control group could be stretched one to two times their original length, while the heat-treated groups could be stretched up to two to three times their initial length until rupture (Fig. 4C).

FIG. 4.

The mechanical properties of PRF membranes after various heat treatments. (A) The stress force of PRF membranes after various heat treatments. (B) The tensile strain at break of PRF membranes following various heat treatments. (C) Representative stress-strain curves of PRF membranes following various heat treatments. Statistical differences were compared between the control nonheated PRF group and the other heating groups. ns: not statistically significant versus control group; *p < 0.05 and **p < 0.01. Color images are available online.

Evaluation of PRF membrane barrier function

To evaluate the ability of PRF membranes to block the migration of HGFs, HGFs were labeled with Hoechst, which could thereafter be excited by ultraviolet light. Three days after seeding HGFs onto PRF membranes from the different groups, the number of fluorescently labeled cells on the upper and lower surfaces of PRF membranes and the number that had penetrated through the PRF membranes and onto the glass slides beneath PRF membranes were evaluated. The fluorescence microscopic images showed that after seeding, significantly more fluorescently labeled cells remained on the heated surfaces than on the control group.

More cells were also found on the bottom glass slides in the control nonheated group, which confirms that more HGFs were able to migrate through regular PRF membranes. Thus, this experiment confirms that the heating of PRF membranes can improve their barrier function (Figs. 5 and 6).

FIG. 5.

Effect of PRF heat treatment on HGFs viability. Hoechst-stained HGF is shown in blue. (A–D) Upper surface of PRF. (E–H) Bottom surface of PRF. (I–L) Cross section of PRF. (M–P) Note that heating disrupted the viability of cells. HGF, human gingival fibroblast. Color images are available online.

FIG. 6.

Quantitative results from the Hoechst-stained HGFs. (A) Upper surface of PRF. (B) Lower of PRF. (C) The cells that passed through PRM membrane and reached the glass slides below the membrane. Statistical differences were compared between the control nonheated PRF group and the other heating groups. ns: not statistically significant versus control group; **p < 0.01 and ***p < 0.001. Color images are available online.

Influence of PRF membranes after heat treatment on human osteoblast proliferation

Human osteoblasts were then cultured with 20% conditioned media from the various PRF groups. CCK-8 assays were performed at 1, 3, and 5 days to quantify cell proliferation. Osteoblasts were mainly attached to the bottom of the 96-well plates 1 day after colonization and have not proliferated in a large amount. The results of CCK-8 on the first day did not show significant differences between different groups, which may reflect that the initial cells' distribution was relatively even.

It was found that the control nonheated group induced significantly higher cell numbers at 5 days compared to that of either heat treatment group, while the single-sided group had a higher proliferation rate than that of the double-sided group (Fig. 7). It is noteworthy to point out that even the double-sided heat treatment group still demonstrated a proliferative increase in cell numbers throughout the entire time points evaluated, confirming remaining bioactivity ever after thermal manipulation of PRF membranes.

FIG. 7.

The proliferation capacity of human osteoblasts cultured with 20% conditioned medium from PRF samples following heat treatment. CCK-8 assays were performed at 1, 3, and 5 days postseeding. *p < 0.05, **p < 0.01, and ***p < 0.001. Color images are available online.

Degradability of PRF membranes

The degradation of PRF membranes was then examined and recorded on a daily basis. The control PRF membranes degraded significantly faster than the heated groups, with complete degradation in all membranes in 2 weeks. In comparison, only between 1/3 and 1/2 of the heat-treated PRF membranes were degraded (Fig. 8). The PRF membranes in the single-sided heat treatment group were degraded in ∼3 weeks, while the PRF membranes using the double-sided heat treatment degraded after 3 to 4 weeks of incubation.

FIG. 8.

(A) Photos and (B) quantification of the time-course degradation of PRF membranes following heat treatment. The degradation of PRF samples was examined in Dulbecco's modified Eagle's medium with 10% FCS and 1% antibiotics (Gibco, Thermo Fisher Scientific). Each group was analyzed with three replicates. Statistical differences were compared between the control nonheated PRF group and the other heating groups. *p < 0.05, **p < 0.01. FCS, fetal calf serum. Color images are available online.

Discussion

For the successful use of GBR, the most important aspect surrounding this concept is to prevent the fast-growing soft tissue from growing into the bone defect before its formation. Ideal barrier membranes applied in GBR should therefore demonstrate good spatial stability, ideal biocompatibility, and cellular blockage of the epithelial tissue. It has previously been reported that the ideal barrier membrane should remain in the defect area for a minimum of 3–4 weeks to provide stable space maintenance necessary for bone tissue ingrowth.³⁸ Compared to other methods to improve collagen membrane stability, such as crosslinking by adding glutaraldehyde or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, thermal manipulation of PRF prevents any potential foreign body reaction and is easily harvested from a peripheral vein at low cost.³⁹

Consistent with the results of previous studies, the degradation time of PRF membranes can be extended by heating.³² Our research results indicated that the size and weight of PRF membranes were reduced following heating and that the porosity of the fibrin mesh was reduced, as observed by SEM. During the heating process, the secondary structure of the albumin protein was modified, and new hydrogen and ligations were formed, which might contribute to reducing the penetration of incoming cells, thereby reducing its degradation rate and improving its stability.³⁹

Prolonging the preservation of the PRF at the implantation site allows it to serve as a more clinically reasonable GBR membrane. Nonetheless, the changes in PRF membrane properties after heating, including cell activity, penetration ability, and the effect on cells, have not previously been studied in detail.

Combined with the previous temperature of heating from previous studies,³² we proposed two heating options, a single-side only and a double-side heat treatment method. Thereafter, the characteristics and barrier function of PRF membranes were investigated. SEM of the nonheated surface of the single-sided heating membranes showed that the fibrous protein structure remained relatively uniform and standard in structure when compared to the heated surface. Analogous to the routine clinical application of collagen membranes, a denser and smoother surface is used adjacent to the soft tissue to block gingival fibroblasts, while a loose and rougher surface is utilized on the bony side of the defect to promote osteoblast growth.

In subsequent barrier experiments, we found that the double-sided heated PRF membrane could prevent more cells from passing through to a greater degree; however, the viable cells were compromised. Degradation experiments also showed that the double-sided heated membrane was also improved, which again signifies that it may be better suited to maintain the space of the bone defect for longer periods of time during clinical applications. Thus, clinical procedures may be optimized based on this knowledge. For instance, perhaps for small horizontal augmentations, single-sided heat treatment would suffice, leading to optimal healing of the defect since more cell viability/activity and growth factors would be improved.

In other scenarios, such as for larger vertical GBR procedures or even to cover titanium meshes, long-lasting double-sided heat-treated membranes would be preferred. Thus, it is clear that future animal and clinical research is needed, but this offers a new and exciting treatment modality whereby membranes that are longer lasting can be fabricated using 100% autologous sources. This has never been possible in the past.

This offers clear advantages over collagen in that not only is the preparation process of lower cost but also it incorporates living cells and growth factors beneficial for wound healing, which is not currently the case when utilizing collagen membranes. Although thermal manipulation can quickly increase the strength and prolong the degradation time of PRF membranes without additives, it also leads to cell damage due to high temperatures and therefore also weakens the biological advantages of PRF. While we did find that the single-side heat treatment group retained more viable cells, future research in this space could prove beneficial.

This research may also generate new ideas for future clinical applications. For example, it is known that mesh exposure is a common complication when using titanium meshes for ridge augmentation.^40,41 Currently, many clinicians are utilizing PRF to cover the titanium mesh with the aim of minimizing the rate of mesh exposure. The data from this study indicate that heat-treated PRF membranes with stronger mechanical properties and longer degradation times would favor more desirable clinical outcomes. Future research to investigate these open questions is therefore needed.

Conclusion

Our study demonstrated that heat treatment of PRF membranes exhibited markedly better mechanical properties than control standard PRF membranes. Heating reduced the number of living cells and fibrin pores in the PRF membranes, which resulted in an increased barrier function. Nevertheless, heating reduced the number of active cells in PRF, which reduced its bioactivity. The single-side heating method not only had the possibility of improving the mechanical properties and degradation performance of the membrane but also retained more living cells and cell activity. In a clinical setting, this membrane could be selected with the outer membrane in contact with the epithelial tissue, acting as the barrier, whereas the interior is more bioactive to further help with new bone formation.

Furthermore, treatment methods could be selected according to the needs of the clinician to achieve the most ideal membrane. Moreover, heating brings about a new feature to extend the resorption properties of PRF to those observed with routine collagen membranes utilized frequently in GBR procedures, with the added advantages of being 100% autogenous and containing living cells capable of releasing growth factors with added bioactive potential.

Footnotes

Authors' Contributions

S.Y. and Y.W. contributed equally. S.Y., Y.W., and Y.Z. conceived and designed the research. S.Y. performed the experiments. S.Y. and Y.W. performed the analysis, interpretation, and wrote the article. S.Y., Y.W., R.J.M., and Y.Z. critically revised the article. All authors approved and agreed to the final revision.

Data Availability Statement

The data that support the findings of this study are openly available.

Ethics Approval Statement

Patient Consent Statement

All participants provided informed consent before treatment on the study.

Disclosure Statement

No competing financial interests exist.

Funding Information

This work was supported by the National Natural Science Foundation of China (81771050) and The National Science Fund for Distinguished Young Scholars (82025011). This work has not received any specific grant from a business.

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