Thermal Manipulation of Human Bone Collagen Membrane (SoftBone) and Platelet-Rich Fibrin (PRF) Membranes

Abstract

Resorbable barrier membranes, including platelet-rich fibrin (PRF) and collagen membranes, can play a key role in guided bone regeneration surgeries (GBR) in dentistry. A new collagen membrane made of partially decalcified allogeneic cortical bone, termed SoftBone membrane (SB), was produced by West Hungarian Regional Tissue Bank. It can be easily adapted to diverse surfaces. Fresh and freeze-dried folded-PRF membranes were compared with freeze-dried SB. Important properties of membranes were reported (moisture content, rehydration capacity, and resistance against proteolytic enzyme). The SB exhibited the best resistance against enzymatic digestion on day 21, its weight was 34% of the original. Fresh F-PRF (folded PRF) disintegrated on the 11th day, while the freeze-dried F-PRF membrane dissolved completely on day 8. The thermal manipulation of the F-PRF membrane using freeze-drying has advantages and also disadvantages in comparison to the fresh one.

1. Introduction

Collagen membranes have been utilized for decades in numerous surgical fields, including neurosurgery, abdominal surgery, othorhinolaryngology, and dental surgery [1, 2, 3, 4, 5].

GBR is a popular surgical process for bone augmentation before dental implantation. There were different collagen membranes historically but in recent times resorbable membranes have been mostly applied. During the resorption period, these barrier membranes can protect the submembranous bone particles from the unintended ingrowth of fibroblasts and epithelial cells (see Figure 1).

Figure 1.

Clinical application of barrier membrane (blue) in GBR. The membrane can protect the grafted bone particles from the unintended ingrowth of gingival fibroblasts and epithelial cells.

Therefore bone morphogenesis can remain intact, but it can be impacted by several other factors, such as local pressure and infection. Bovine xenogeneic membranes and allogeneic membranes can also be found on the market. Their main advantage is the strong biomechanical properties and the ability for resorption [5, 6, 7].

The main preservation method of the collagen membranes is freeze-drying (lyophilization). The aim of this process is to decrease the water content of the tissue by under 5% so that these freeze-dried products can be stored at room temperature. Without water the autolytic enzymes cannot work, so membranes remain intact. Before or during surgery the membrane is rehydrated.

During the last two decades, the number of reports on the clinical application of different platelet-rich products in oral surgery and implant dentistry has increased. Especially those examining local tissue regeneration and include GBR either alone or in combination with particulated bone grafts. Several techniques for platelet concentrates have been introduced in the surgical field for the acceleration of tissue regeneration. Two main groups of platelet-rich products can be distinguished—(1) first-generation platelet-rich plasma (PRP) products with anticoagulants and (2) second-generation PRF products without anticoagulants. PRF can also be used as a barrier membrane in GBR similar to a collagen membrane [8, 9, 10, 11].

The main effect of platelet-rich products is based on the theory of regenerative properties of the autologous cells, such as leukocytes (LCs), platelets (PLs), and stem cells. They release cytokines and growth factors (e.g., PDGF-platelet derived growth factor, EGF-epithelial growth factor, VEGF, and TGF-transforming growth factor) for 2–3 weeks in vitro playing a crucial role in bone and soft tissue regeneration. A total of 10⁶/μl platelets are likely and are in the therapeutically effective range; it is 4–5 times higher than baseline platelet count values in whole blood [12].

PRF is a special mixture of individual cells entrapped in a freshly nascent fibrin clot that later serves as autoscaffold. Depending on the leukocyte or fibrin content, platelet concentrates can be classified into different categories—leukocyte and platelet-rich plasma (L-PRP); pure platelet-rich fibrin (P-PRF); leukocyte and platelet-rich fibrin (L-PRF); F-PRF; injectable i-PRF; advanced A-PRF; autologous albumin gel and platelet-rich fibrin (Alb-PRF); etc. [13, 14, 15, 16, 17, 18, 19, 20, 21, 22]. Unfortunately, the names of different membranes are sometimes confusing.

The F-PRF membrane was created and published by our group [20]. It has many attractive properties (e.g., homogenous cell distribution and better biomechanical properties) in comparison to conventional methods.

Many articles describe these different PRF membranes. Their properties that have been reported include platelet number, cell number and their distribution in membranes, biomechanical properties, and cytokine distribution and their release over time. This report is not going to recapitulate these findings. Generally, PRF membranes are prepared at the bedside for immediate autologous clinical application and short-term use (for up to 14-days) but this approach somewhat limits their therapeutic value [16, 17, 18, 19, 20, 21, 22, 23].

PRF membranes accelerate bone substitute healing and allow for earlier implant placement compared to collagen membranes, but collagen membranes can stimulate bone regeneration significantly during later stages, as was reported in a model using sheep [24].

Metabolic activity and proliferation of human osteoblast cells in vitro were supported to a significantly higher extent by eluates from fresh PRF membranes. Collagen and PRF membranes are suitable as scaffolds for the cultivation of human osteoblast cells in vitro; proliferation was significantly higher on PRF membranes and PRF clots than on BioGide® collagen membranes [25].

The PRF membrane can be applied as a sole barrier membrane only when re-entry (a second surgical flap) is not clinically required. In extensive GBR cases, PRF membranes are combined with either a collagen barrier membrane or titanium mesh. As a rule of thumb, it is always advantageous to utilize PRF on the outer surface of GBR procedures over top the collagen barrier membranes. There are two reasons for this—a) if PRF is left exposed to the oral cavity, its high quantity of pathogen-fighting leukocytes dramatically reduces the chance of infection (nearly tenfold); b) PRF is known to rapidly promote greater soft-tissue wound healing compared to hard tissues [11].

During the last decade, many articles focused on freeze-dried platelet products, including PRF membrane. It was reported that the new bone formation in the fresh/lyophilized PRF (1:1) was much more than that of other groups both 6 and 12 weeks in rabbits. The data suggested that growth factor concentration and release kinetics are a consequence of fresh and lyophilized PRF combination, which is an effective way to promote bone regeneration [26].

Lyophilization offers storage and processing benefits over conventional methods including longer storage time at room temperature and rapid transformation by rehydration that enables the practical application in emergency medicine and increased stability for transport. The combination of platelet-rich products and biomaterials has increased the therapeutic value of this biomaterial. PRF can bridge the gap between scaffolds and cell biology, adding the biological stimulus required for functional tissue regeneration [27].

According to some reports, freeze-drying of platelets can provide promising results and enhance the properties of the PRF membrane. Freeze-dried PRFs express their growth factors more slowly than intact ones. They have a favorable effect on bone morphogenesis and these platelets cause elevated Runt-related transcription factor 2 (Runx2) in alveolar bone and a 10-fold rise of alkaline phosphatase levels and mineralization factors. A 1.6-fold increase in osteoblast proliferation was also reported when compared to fresh PRF. A total of 97% bony coverage was detected in a rat craniofacial defect model compared to 84% for fresh PRF. The cell viability of the PRF membrane is not such an important issue in long-term bone tissue regeneration. Freeze-dried PRF, as a biomimetic scaffold without living cells, showed better biological properties in osteoblast and mesenchymal stem cell colonization and proliferation in vitro and animal studies as well. The possible explanations for this surprising phenomenon are increased pore size in freeze-dried PRF, which provides an ideal condition for cell adhesion and improved release of intact growth factors and cytokines [28]. Freeze-dried platelets had a five-fold increase in blood vessel density in a healing wound model with diabetic mice in contrast to the nontreated group [29].

The strengths and limitations of lyophilized platelets were collected in Table 1.

Strength

1. Preservation of biological properties

2. Preservation of morphological architecture

3. Sustained release of growth factors

4. 100% natural and autologous

5. Biocompatible with other biomaterials

6. Multiple usages with single venipuncture

7. Easy transportation

8. Better storage capabilities

9. Enables use in emergency surgery

10. Longer clinical shelf-life

Limitations

1. Fabrication cost

2. Possible risk of contamination

3. Demands standardization protocol for lyophilization technique.

Table 1.

Strengths and limitations of the lyophilized platelet concentrate (LPC) [27, 30].

In spite of many articles in this field, our knowledge is still not consistent enough. There are some uncertainties due to graft properties, for example, platelet number, etc., so the processes can be standardized but the products cannot.

These membranes are prepared in centrifuge tubes alone or with metal kits. In spite of a large amount of research on this, a routine significant amount of important information is still missing that concerns the ideal preparation of the product in daily practice.

An “ideal” barrier membrane should present the following characteristics [5, 31].

• biocompatibility (to prevent adverse reactions with the surrounding tissue and with the organism);

• tissue integration (to favor the embedding in the surrounding tissue and allowing a progressive integration of collagen fibers);

• dimensional stability (the positioning and shape of the membrane should remain unaltered till degradation);

• handling (the membrane should be managed and easily placed over the defect);

• selective permeability (the membrane should be able to exclude unwanted epithelial cells while allowing osteogenic cells to proliferate);

• space making function (to provide space for a stable blood clot, to allow bone regeneration).

The goal of the report is to characterize some properties of the newly invented freeze-dried human collagen membrane made of cortical bone termed SB and folded platelet-rich fibrin (F-PRF) membranes after thermal manipulation and freeze-drying. Subsequent enzymatic digestion in cell culture conditions mimics the rigor of clinical conditions. Additionally, the resistance of collagen and PRF membranes was compared.

2. Materials and methods

2.1 Preparation of collagen membrane from human cortical bone (patent pending SB by the Hisztolabor Ltd and Dent-Art-Technik, Győr, Hungary)

A 28-years old tissue donor’s femoral diaphysis was processed. Cortical plates with a size of 15 × 10 × 0.5 mm were cut using a buzz saw with a diamond edge. Ten cortical plates were processed using typical bone processing, which includes defatting, decellularization, and partial decalcification. The membranes were freeze-dried in a ScanVac Superior Pro freeze-dryer (Labogene, Denmark).

2.2 Folded and non-folded PRF membrane preparation

1. Blood collection: blood samples were collected with the informed consent of three donors. Experiments were in accordance with the ethical standards and approval of the Regional Research and Ethics Committee. Whole blood was drawn by venipuncture from cubital veins into 36 nine-ml vacutainer tubes without any chemicals (Vacuette, Gerner BioOne).

2. Cell separation: due to the short lifespan of some cytokines, growth factors, and clotting of the blood sample, cell separation had to start as soon as possible, within 2 minutes maximum as previously reported [20]. Cell separation was performed by Steinberg centrifuge (CGOLDENWALL 80–2) at 375 revolutions per minute (RPM) for 10 minutes to prepare F-PRF. ~4 ml of plasma could be removed from each tube, and 16–18 ml plasma was put into eight rectangular metal jars manufactured for PRF (Dent-Art-Technik Ltd., Győr, Hungary). After 8 min at room temperature, fibrin filaments started to appear.

3. Folded PRF preparation: in four PRF jars in the early gelatinous stage the membrane was folded 4–5 times with forceps by seizing the corners of the membrane to completely enmesh and entrap the blood cells within the dense fibrin network (see Figure 2a). The membrane could be formed into different shapes by squeezing out the fluids present in the fibrin clot using a stainless steel compression device. The membranes were cut into uniform pieces and weighed.

4. Unfolded, intact PRF preparation: in four PRF jars the plasma was left intact until there was clotting and spontaneous formation of the flat rectangular membrane. The membranes were cut into uniform pieces and weighed.

Figure 2.

(a) Preparation of folded PRF and (b) freeze-dried F-PRF. Note cracked surfaces.

2.3 Freeze-drying of F-PRF membranes

The same procedure was performed as in the SB membrane (see #2.1 and Figure 2B).

2.4 Determination of moisture content after FD by gravimetry

Samples of freeze-dried F-PRF and SB collagen membrane (five pieces from each group) were put into a drying chamber (Binder, Germany) for 1 h at 90°C to remove the remnant moisture of lyophilized tissue and assess the efficacy of FD. The tissue samples were weighed after freeze-drying and heat drying as well.

2.5 Rehydration

All membrane pieces of the three groups (SB collagen membrane, folded PRF, and intact PRF) were put in rehydration fluid. Half of them were submerged into a PS, the other half were rehydrated in the blood donor’s serum. According to a previous experiment during 10 min of rehydration, all the membranes were saturated with moisture and their weight did not change over the next 20 min of rehydration.

After freeze-drying, the pieces were weighed. According to our previous report, the F-PRF is superior to a conventional non-folded membrane, so only fresh and lyophilized rehydrated F-PRF and lyophilized SB membrane were investigated in the following #6 experiment [20]. Unpaired two-sample t-tests were performed to compare the differences of the relevant experimental groups.

2.6 Membrane resistance against proteolytic digestion

The objective of this experiment was to mimic the clinical conditions and assess the resistance of membranes against enzymatic protein digestion after transplantation. Three groups (freeze-dried SB collagen, freeze-dried and rehydrated F-PRF, fresh F-PRF) were prepared by cutting them into uniform pieces. All fresh, freeze-dried and rehydrated membranes were weighed then kept in Dulbecco’s minimal essential medium (DMEM) containing 0.1% (v/v) trypsin (Sigma, USA) and 0.4 mg/ml EDTA (ethylene diamine tetraacetic acid) at cell culture conditions in a HeraCell 150 CO₂ thermostat (Heraeus, Germany) at +37°C in 5% CO₂ to feed the membranes. DMEM with trypsin was replaced every day. The weights of membrane pieces were assessed every 24 h using a laboratory scale (Kern, Germany) for 21 days. The samples were kept in a 2-ml mixture in a 24 cell well plate.

2.7 Histology

HE, trichrome, and immunohistological staining (VEGF) were performed in the fresh F-PRF and the SB groups.

3. Results

The results of thermal manipulations can be seen in Table 2. The SB membrane lost more than 50% weight during FD. The folded PRF membrane lost less water than the non-folded membrane in percentage, and both membranes became fragile after freeze-drying (see Figure 2b).

Weight after freeze drying
Folded PRF (n = 15)		16.2 ± 2.2%
Non folded PRF (n = 15)		10.3 ± 4.1%
SB (n = 10)		43 ± 0.4%
Weight after rehydration (10 min)
F-PRF			Non folded PRF
PS (n = 5)	serum (n = 5)		PS (n = 5)	serum (n = 5)
52.4 ± 4.8%	48.4 ± 2.4%		30 ± 6.8%	30.8 ± 7.2%
SB (n = 5; in PS only): 66 ± 0.3%
Moisture remnant after freeze-drying:
F-PRF (n = 5): 3.2 ± 0.3%
Non folded-PRF (n = 5): 3.3 ± 0.3%
SB (n = 5): 3.9 ± 0.4%

Table 2.

Comparison of weights after freeze drying and rehydration process. The original fresh weight of the PRF membranes was set at 100%. The processed decalcified weight was set at 100% in SB. (PS-physiological salt solution).

The difference between the rehydration capacity of folded and non-folded PRF membranes is significant (p < 0.01). No difference was found between PS and blood serum after rehydration. The SB membrane regained 66% of the original weight (see Table 2).

The moisture content was under 5% in both PRF groups and in SB as well after FD.

There was a remarkable resistance against enzymatic digestion for 1 week in fresh F-PRF. During the 2nd week, dissolving was accelerated. The fresh F-PRF was obviously superior to the freeze-dried F-PRF, which disintegrated on the 8th day. The SB collagen membrane had the best resistance. Until the 21st day, it preserved 34% of the original rehydrated weight (see Figure 3).

Figure 3.

Proteolytic digestion in membranes. Weights were expressed in relative %. On day 0 the rehydrated weight was set at 100% in freeze-dried PRF and SB membranes. Bars are standard deviations.

Histology and immunohistology: the F-PRF contains many cells in homogenous distribution, especially leukocytes and platelets in a dense fibrin network (Figure 4).

Figure 4.

Histology of fresh F-PRF membrane (HE, 400× magnification). In a fresh F-PRF membrane remarkable cell quantity can be seen. There are big leukocyte groups separated by fibrin bunches (arrow).

Figure 5.

Cloudy brown VEGF (vascular endothelial growth factor) positivity can be seen around dense platelet groups (VEGF immunohistology, 400× magnification). Blue cells are leukocytes. Note the remarkable early phase of vasculogenesis (arrow), which is a key issue in tissue morphogenesis.

Figure 6.

(a) SoftBone after processing (note the pliability). (b) Note the transparent SB membrane and original intact cortical bone plate with holes. (c) Histology: parallel red collagen fibers can be seen in decalcified SB (horizontal arrow). The osteocyte lacuna is empty (vertical arrow). (trichrome staining 400×). (d) SB membrane after 3 weeks of enzymatic digestion. The original osteon units of cortical bone can still be recognized with central Haversian canals. (Trichrome staining 400×).

The SB membrane became pliable and transparent after tissue processing, freeze-drying, and rehydration (see Figure 6a and b). In decalcified and processed bone the collagen was preserved (see Figure 6c). After 3 weeks of enzymatic digestion in spite of remarkable weight loss, the original structure seemed to be intact. The blue color is a sign of molecular tissue disintegration using the same trichrome staining (see Figure 6d).

4. Discussion

During freeze-drying, the F-PRF lost less moisture in comparison to the non-folded one (84% vs. 90%). Squeezing removes more serum in F-PRF than in the spontaneous contraction of fibrin network in non-folded PRF membrane, so the latter had higher initial water content.

After rehydration, the F-PRF could regain more than half of its original weight, while intact PRF reached only 30%. It seems that the folding technique provides advantages in this field too; probably the water binding capacity remained more intact this way. Ten minutes are enough for rehydration, but freeze-dried PRF membrane could absorb almost 30% more additional moisture slowly during the next 24 h (see Figure 3).

There was no significant difference between the PS and blood serum as a rehydrant. The folded and non-folded PRF membranes were rehydrated by solution in the same manner. SB became pliable again after rehydration, so it can be adapted to irregular surfaces.

The SB collagen membrane showed more resistant properties in tissue culture conditions than the F-PRF membrane. The freeze-dried F-PRF could not withstand the double rigor of tissue culture conditions and enzymatic digestion that mimic the in vivo clinical conditions in the oral cavity. The membranes are separated from saliva and oral liquids clinically in optimal conditions, but they are exposed to long-term unfavorable enzymatic and protecting cellular effects. The fresh F-PRF disintegrated after 11 days and the freeze-dried F-PRF membranes dissolved on the 8th day in these conditions. After 3 weeks of storage, the SB membrane still seemed intact macroscopically but its weight fell to 34% of its original.

It seems the folding procedure increases the resistance and density of the fibrin network serving as a biological autoscaffold and showed better resistance against enzymatic digestion of membrane proteins in fresh F-PRF. In contrast to the fresh membrane, freeze-drying decreased the resistance of the F-PRF membrane so its disintegration was faster.

The biological value of fresh F-PRF membranes is remarkable in that it contains homogenously distributed active living cells. The early phase of vasculogenesis can be found after few minutes, which is very astounding and an important milestone on the path to developing blood supply in tissue morphogenesis (see Figures 4 and 5).

The F-PRF process obviously requires manual manipulation, so it needs to be performed under aseptic conditions clinically. After the early introduction of open-system experts were alerted to safety issues [32]. During the last decade, the huge and emerging number of clinical cases did not confirm the worries. There were no available references on serious adverse events (SAE) or serious adverse reactions (SAR) in regard to the application of platelet and leukocyte-rich products.

Freeze-drying as a preservation method can produce well manageable tissue samples and even enhance some favorable properties of PRF.

The resorbable collagen membranes, including SB, do not have such an attractive biological value but they do have their advantages. They serve like a natural protective biological scaffold for tissue morphogenesis. SoftBone is an allogeneic decellularized and decalcified flexible bone membrane, which mostly consists of collagen type I. It provides an optimal condition for host cells to potentially have ideal pore sizes. Its remarkable resistance against enzymatic digestion in vitro makes it a good choice alone or in combination with PRF membrane for long-term protection of grafted bone; however, further studies are required to find the optimal membrane type for GBR.

5. Conclusion

Resorbable barrier membranes, including platelet-rich fibrin (PRF) and collagen membranes, can play a key role in guided bone regeneration surgeries (GBR) in dentistry. PRF membranes have a high biological value containing a lot of cytokines and growth factors. In spite of good reported experimental results of freeze-dried PRF, its enzymatic disintegration was faster than in fresh PRF. SoftBone membrane made of partially decalcified allogeneic cortical bone can be easily adapted to diverse surfaces and showed satisfying resistance against proteolytic trypsin digestion. Further studies will show the long-term fate of SB membrane in animal and clinical studies.

F-PRF and SB membranes alone or in combination have an excellent potential to become ideal membranes in GBR.

Acknowledgments

We would like to express our thanks to Mr. John Kowalchuk for his support in editing the manuscript.

Abbreviations

FD	freeze-drying (lyophilization)
F-PRF	folded platelet-rich fibrin
GBR	guided bone regeneration
HE	hematoxylin–eosin staining
PRF	platelet-rich fibrin
PS	physiological salt solution
SB	SoftBone membrane (patent pending product)
VEGF	vascular endothelial growth factor