In vitro evaluation of mechanical properties of platelet-rich fibrin membrane and scanning electron microscopic examination of its surface characteristics.

BACKGROUND: The aim of this study was to evaluate the mechanical properties of the platelet-rich fibrin (PRF) membrane and to compare these properties with that of commercially available collagen membranes used for guided tissue regeneration (GTR) procedures. Scanning electron microscopic (SEM) examination of PRF membrane was also performed to determine the cell distribution pattern within the different regions of the membrane.
MATERIALS AND METHODS: Modulus of elasticity and hardness of (i) PRF membrane (ii) bovine collagen membrane and (iii) fish collagen membrane were assessed by performing surface indentation test using T1 950 Triboindenter. The in vitro degradation tests were conducted by placing the (i) PRF membrane (ii) bovine collagen membrane and (iii) fish collagen membrane of equal sizes (10 mm × 5 mm) in 5 ml of pH 7.4 phosphate buffer solution on a shaker set at 40 rpm for 1-week. The degradation profiles were expressed as the accumulated weight losses of the membrane. SEM evaluation of the PRF membrane was done under both low and high magnification.
RESULTS: Young's Modulus of elasticity was found to be 0.35 GPa for PRF membrane, 2.74 GPa for bovine collagen membrane and 1.92 GPa for fish collagen. The hardness was 10.67 MPa for PRF membrane, 110.7 MPa for bovine collagen membrane and 90.5 MPa for fish collagen membrane. PRF membrane degraded by about 36% of initial weight after a 1-week in vitro shaking test. Fish collagen membrane degraded by about 8% of initial weight, bovine collagen membrane degraded by about 3% of initial weight. Dense clusters of platelets formed due to extensive aggregation, and few leukocytes were observed in buffy coat area.
CONCLUSIONS: The preliminary findings from the assessment of the mechanical properties of PRF membrane showed that it was lacking in several desired properties when compared to commercially available collagen membranes. Lack of rigidity and faster degradation may limit its application in GTR procedures.

INTRODUCTION

The hallmarks of periodontal disease are destruction of soft connective tissues, bone loss, and loss of connective tissue attachment to cementum; these alterations, if left untreated, lead to tooth loss. Periodontal regeneration is defined as the reproduction or reconstruction of lost or injured tissue, so that the form and function of the lost structures are restored. Guide tissue regeneration (GTR) employs a barrier membrane around the periodontal defect to prevent epithelial down growth and fibroblast transgrowth into the wound space, thereby maintaining a space for true periodontal tissue regeneration.[1] As such, this procedure has been, and still is, widely employed in periodontal clinics and established as a basic technique in periodontal regenerative medicine.

Development of the bioactive surgical additives is one of the great challenges of clinical research which has been used to regulate inflammation and increase the speed of the healing process. In the past two decades, increased understanding of the physiological roles of platelets in wound healing and after tissue injury has led to the idea of using platelets as therapeutic tools.[2] Platelet-rich fibrin (PRF) described by Dohan et al., is a second-generation platelet concentrate consisting of fibrin membrane enriched with platelets and leukocytes.[3] This technique requires neither anticoagulant nor bovine thrombin (or any other gelling agent). It is nothing more than centrifuged blood without any addition. By driving out the fluids trapped in the fibrin matrix, practitioners will obtain very resistant autologous fibrin membranes.[3] Thus, PRF membrane may act as a third-generation membrane with its source of growth factors (GFs) from the cells trapped inside the fibrin matrix. The PRF membrane being autologous in nature and cost-effective compared to any other membrane offers significant advantages over commercially available membranes.

When considering the development of GTR barrier materials, the mechanical properties ought to be studied. In addition, the appropriate degradation rate of the material has to be evaluated to assess whether it meets the requirements and diversities of tissue regeneration procedures.[4] Scanning electron microscopic (SEM) examination of PRF membrane was also performed to determine the cell distribution pattern within the different regions of the membrane. The main objective of this study was to evaluate the mechanical properties like modulus of elasticity, hardness, in vitro degradation of PRF membrane, along with SEM examination of its surface. The secondary objective of this study was to compare these properties with that of commercially available bovine collagen membrane and fish collagen membrane.

MATERIALS AND METHODS

The study was conducted at Hysitron Nanotechnology, Technopark, Trivandrum, India (modulus of elasticity and hardness) and National Institute of Technology, Calicut, India (in vitro degradation and SEM examination) during November 2012.

Preparation of platelet-rich fibrin

Ten milliliter blood was drawn from the investigator (26-year-old male) by venipuncture of the right antecubital vein. Blood was collected in sterile glass test tubes without any anticoagulants and immediately centrifuged on a rapid angiogenesis and an easier remodeling top centrifuge (Micro Centrifuge KW 60, Almicro Intstruments, Haryana, India) at 3000 rpm for 10 min. This resulted in the separation of three basic fractions because of differential densities: The bottom red blood cells (RBCs), middle PRF gel, and the top layer of platelet-poor plasma (PPP). PPP was aspirated and discarded, and the PRF gel was separated from underlying RBCs by the use of sterile stainless steel scissors. PRF membrane was prepared by pressing the PRF gel between two pieces of surgical gauze [Figure 1].

Figure 1

Preparation of platelet-rich fibrin membrane

Evaluation of mechanical properties

The basic properties of PRF membrane were evaluated by a series of tests to determine its modulus of elasticity, hardness and in vitro degradation.

Modulus of elasticity and hardness

The membrane was cut into 10 mm × 5 mm strips and hydrated with ph 7.4 phosphate buffer solution (PBS) before being subjected to mechanical testing. The modulus of elasticity and hardness of the PRF membrane, bovine collagen (Healiguide, EnColl, Freemont, CA, US) and fish collagen (Periocol, EucarePharmaceuticals, Chennai) membrane were recorded by performing surface indentation test with T1 950 Triboindenter (T1 950 Triboindenter, Hysitron Nanotechnology, Minneapolis, US) at <1 uN load [Figure 2]. A total of five readings from different locations for each membrane were recorded, and the mean value was computed.

Figure 2

T1 950 Tribolndenter surface indenter

In vitro degradation

The in vitro degradation tests of the prepared membranes were conducted by placing the PRF membrane of size 10 × 5 mm in 5 ml of pH 7.4 PBS on a shaker set (Orbital KAHN Shaker, Lab Line Instruments, Kochi, Kerala, India) at 40 rpm. Strips of similar sizes (10 mm × 5 mm) of commercially available barrier membrane (bovine collagen and fish collagen) were placed separately on the same shaker set to compare the in vitro degradation profiles [Figure 3]. All the three membranes were weighed on an electronic micro weighing scale and at the end of 1-week, the membranes were taken out of the incubation medium, washed with distilled water, dried, and its weight was measured. The degradation profiles were expressed as the accumulated weight losses of the membrane.

Figure 3

Shaker set for assessing in vitro degradation

Scanning electron microscopic observation

The surface microstructure of membrane was examined by SEM (FESEM, SU6600, Hitachi High Technologies America, Inc.). Before SEM observation, the sample was dehydrated by passing the specimens through a graded series of ethanol-water mixtures, and then dried by the critical-point method. After drying the sample was sputter-coated with gold, and examined under an SEM. Two areas were scanned, (i) Mid membrane region and (ii) the junction between the red part and the yellow part of the fibrin clot (buffy coat area).

RESULTS

Modulus of elasticity and hardness

A total of five readings from different locations for each membrane were recorded, and the mean value was computed. When the PRF membrane was compared with a commercially available collagen membrane (bovine collagen and fish collagen), it was found that their mechanical properties were lower in terms of modulus of elasticity and hardness. Young's Modulus of elasticity was found to be 0.35 GPa for PRF membrane, while it was 2.74 GPa for bovine collagen membrane and 1.92 GPa for fish collagen. The hardness was 10.67 MPa for PRF membrane, 110.7 MPa for bovine collagen membrane and 90.5 MPa for fish collagen membrane [Table 1].

Table 1

Recordings for modulus of elasticity and hardness

In vitro degradation

The PRF membrane was found to have maintained its physical form up to 6 days, at the end of 1-week, the membrane showed a considerable amount of degradation. Fish collagen membrane also showed signs of degradation (1-week), but the bovine collagen showed only very mild signs of degradation (1-week). The membrane degradation test result is shown in Graph 1. PRF membrane degraded by about 36% of initial weight after a 1-week in vitro shaking test. Fish collagen membrane degraded by about 8% of initial weight, and bovine collagen membrane degraded by about 3% of initial weight [Graph 1].

Graph 1

Results of 1-week degradation test of platelet-rich fibrin membrane, bovine collagen and fish collagen

Scanning electron microscope evaluation

Platelet-rich fibrin membrane was evaluated under SEM to visualize its surface morphology and the cell types that were trapped within it. Under low magnification of the mid membrane region (250 SE), the surface was found to be highly irregular [Figure 4]. At higher magnification (3000 SE), the junction between the red part and the yellow part of the fibrin clot (buffy coat area) showed spherical structures with an irregular surface which can possibly be identified as leukocytes [Figure 5]. Surrounding it, there was a dense aggregate of activated platelets resting on a mature fibrin background. Beyond the buffy coat area, in the mid membrane region, there were many areas of dense clusters of platelets formed due to extensive aggregation and clotting [Figure 6]. Platelet morphology was totally modified by aggregation and clotting processes. Therefore, it was not possible to identify nonactivated platelets, (discoid bodies) but rather only a large aggregate of platelet-fibrin polymers.

Figure 4

Scanning electron microscopic image at low magnification (250 SE)

Figure 5

Scanning electron microscopic image at high magnification (3000 SE) of buffy coat area, spherical structures identified as leukocytes

Figure 6

Scanning electron microscopic image beyond the buffy coat area (300 SE)

DISCUSSION

The rationale behind the use of PRF membrane lies in the fact that the platelet α granules are a reservoir of many GFs that are known to play a crucial role in hard and soft tissue repair mechanism. These include platelet-derived GFs, transforming GF-β, vascular endothelial GF, and epidermal GF.[5] PRF has to be considered as a fibrin biomaterial. Its molecular structure with low thrombin concentration is an optimal matrix for migration of endothelial cells and fibroblasts. It permits a rapid angiogenesis and an easier remodeling of fibrin in a more resistant connective tissue.[6]

Choukroun and his associates were amongst the pioneers for using PRF protocol in oral and maxillofacial surgery to improve bone healing in implant dentistry. Several studies have examined the effectiveness of PRF in intrabony defects and Grade II furcation defects and have found positive clinical and radiographic outcomes.[7891011] The addition of PRF as a membrane to coronally advanced flap showed an increase in width of keratinized gingiva.[1213] In view of these positive findings, there is a need to extend the usefulness of PRF membrane to GTR applications as well. The assessment of the mechanical properties of PRF membrane for GTR procedures is required before its clinical use.

The modulus of elasticity and hardness were compared with bovine collagen and fish collagen. It was found that the modulus of elasticity and hardness were less for PRF membrane when compared to collagen membranes. This is because PRF membrane is an autologous membrane with no external additives to enhance the physical properties other than the natural resistance offered by the fibrin matrix. The major disadvantage of PRF membrane is its lack of rigidity, because of which the membrane may tend to collapse over the bone and root surface limiting the space, which is necessary for clot maturation. Further research is needed to improve the rigidity of PRF membrane.

The structural integrity of the implanted bio absorbable barrier membrane should be preserved for a sufficient time to ensure desired results. The degradation rate of PRF in vitro was compared with that of two other commercially available collagen membranes to obtain a gross assessment of its degradation profile. The membrane degradation test results showed that PRF membrane was comparable to other membranes in terms of maintaining its physical property up to 6 days. At the end of 1-week, PRF membrane was found to have degraded to about 36% of initial weight, whereas fish collagen to about 8%, and bovine collagen to about 3%. These results however cannot be extrapolated with clinical data because the resorption process could be further facilitated by enzyme digestion in real applications. Future studies to determine the actual degradation of PRF membrane in actual clinical conditions needs to be done to obtain more clinically useful data.

Platelet-rich fibrin membrane was evaluated under SEM to visualize its surface morphology and the cell types that are trapped within it in order to better understand its biologic properties. The junction between the red part and the yellow part of the fibrin clot (buffy coat area) showed spherical structures with an irregular surface which can possibly be identified as leukocytes. There was also a dense aggregate of activated platelets resting on a mature fibrin background in the buffy coat area. Beyond the buffy coat area there were many areas of dense clusters of platelets formed due to extensive aggregation and clotting. Platelet morphology was totally modified by aggregation and clotting processes. Therefore, it was not possible to identify nonactivated platelets (discoid bodies), but rather only a large aggregate of platelet-fibrin polymers. Kawasaki et al., obtained the same results with thrombin-activated PRP and showed the contribution of platelets to the structural rigidity of the fibrin network.[14] The dense clusters of platelets seen along the buffy coat area and yellow part of the membrane confirms the trapping of platelets along the fibrin clot, which is beneficial for wound healing process as they can be a rich source of GFs. Our finding were similar to the findings of Dohan et al., who also reported with increasing numbers of platelets and leukocytes along the buffy coat area.[15] It may be necessary to preserve a small RBC layer at the PRF clot end to collect as many platelets and leukocytes as possible. SEM examination of the PRF-membrane also showed that the fibrin strands were condensed and stuck to each other, which may be due to the effect of compression on the fibrin matrix.

Further in vitro and in vivo studies are needed to evaluate the mechanical properties and to improve the rigidity of PRF membrane. The degradation profile of PRF membrane in vivo should also be assessed to know the exact time up to which PRF membrane maintains its structural integrity to act as an effective barrier membrane.

CONCLUSIONS

The ease of preparation and cost-effectiveness of PRF membrane offers a huge advantage over other commercially available membranes. The preliminary findings from the assessment of its mechanical properties and its comparison with other collagen membranes do not offer promising results. Lack of rigidity and faster degradation may limit its application in GTR procedures. PRF can be considered a healing biomaterial that can be utilized in regenerative surgical procedures to fasten healing, but its application as a barrier membrane is doubtful due to its poor mechanical properties. Autologous PRF membrane, a GF delivery system may offers a promise in the field of periodontal regeneration if it meets the requirements and diversities of tissue regeneration procedures by improving its mechanical properties.