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Keywords:

  • Platelet-rich fibrin;
  • periodontal ligament fibroblasts;
  • osteoprotegerin;
  • alkaline phosphatase;
  • periodontal infrabony defects

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

Background:  Platelet-rich fibrin (PRF) by Choukroun’s technique is derived from an autogenous preparation of concentrated platelets. Little is known about the effects of PRF on periodontal ligament fibroblasts (PDLFs) and the application of PRF for periodontal regeneration.

Methods:  PDLFs were derived from healthy individuals undergoing extraction for orthodontic reasons. Blood collection was carried out from healthy volunteers. PRF was obtained from a table centrifuge centrifuged at 3000 rpm for 12 minutes. The effects of PRF on PDLFs were determined by measuring the expression of phosphorylated extracellular signal-regulated protein kinase (p-ERK), osteoprotegerin (OPG) and alkaline phosphatase (ALP) activity. Moreover, we retrospectively examined the feasibility and safety of reconstructing the periodontal infrabony defects with PRF in six patients.

Results:  PRF was found to increase ERK phosphorylation and OPG in PDLFs in a time-dependent manner (p < 0.05). ALP activity was also significantly upregulated by PRF (p < 0.05). Application of PRF in infrabony defects exhibited pocket reduction and clinical attachment gain after six months. Periapical radiography revealed radiographic defect filled in grafted teeth.

Conclusions:  The enhancement of p-ERK, OPG and ALP expression by PRF may provide benefits for periodontal regeneration. Clinical and radiologic analysis showed that the use of PRF is an effective modality for periodontal infrabony defects.


Abbreviations and acronyms:
ALP

alkaline phosphatase

ANOVA

analysis of variance

BSA

bovine serum albumin

DMEM

Dulbecco’s Modification of Eagle’s Medium

CEJ

cemento-enamel junction

ERK

extracellular sign-regulated protein kinase

FCS

foetal calf serum

OD

optical density

OPG

osteoprotegerin

PDGF

platelet-derived growth factor

PDLF

periodontal ligament fibroblasts

PRF

platelet-rich fibrin

TGF

transforming growth factor

RANKL

receptor activator of nuclear factor-B ligand

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

Platelet-rich fibrin (PRF) described by Choukroun et al.1 is a second generation platelet concentrate which allows one to obtain fibrin membranes enriched with platelets and growth factors, after starting from an anticoagulant-free blood harvest.2,3 PRF looks like a fibrin network and leads to more efficient cell migration and proliferation, and thus cicatrization.4 This unique structure may act as a vehicle for carrying cells that are essential for tissue regeneration. In addition, the PRF membrane could serve as a resorbable membrane for guided tissue regeneration.

Many growth factors, such as platelet-derived growth factor (PDGF) and transforming growth factor (TGF-β), are released from PRF.2,3,5 Recently, studies have demonstrated that the PRF membrane has a very significant slow sustained release of key growth factors for at least one week3 and up to 28 days,6 which means that the membrane stimulates its environment for a significant time during wound healing. Some clinical applications have been described in oral surgery2,3 and implant dentistry.7,8 However, there are few references in the literature about the biological properties of PRF for periodontal infrabony defects.

One of the most dominant factors in the regeneration of periodontal tissues are periodontal ligament cells. Human periodontal ligament fibroblasts (PDLFs), which play an important role in alveolar bone formation and resorption in the development of periodontitis, form a heterogenous population, with some cells having osteoblast-like characteristics and the potential to differentiate into osteoblasts.9 There is also strong alkaline phosphatase (ALP) activity in PDLFs, which appears to be quite important for the apposition of acellular cementum.10

Recently, PRF was found to stimulate the proliferation of human PDLFs.11 However, the underlying mechanisms are not yet completely understood. The extracellular signal-regulated protein kinase (ERK) signalling pathway is one of the mitogen-activated protein kinase cascades and plays an important role in the regulation of cell growth and differentiation.12 Osteoprotegerin (OPG), a naturally occurring inhibitor of osteoclast differentiation, binds to the receptor activator of nuclear factor-B ligand (RANKL) and blocks RANKL from interacting with RANK.13 ALP is one of the markers of osteoblastic differentiation. The aim of this study was to determine whether PRF could influence the functions of PDLFs. The effects of PRF on human PDLFs were determined by measuring protein expression of p-ERK, OPG and ALP. Moreover, we retrospectively examined the feasibility and safety of reconstructing the periodontal infrabony defects with PRF in six patients. This clinical trial was to evaluate the effectiveness of PRF in promoting pocket depth reduction, clinical attachment gain and bony defects filled in grafted teeth.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

PRF preparation

The human blood samples in this study were obtained with the permission of the Institutional Review Board at Chung Shan Medical University Hospital, Taichung, Taiwan. Blood from 10 non-smoking healthy volunteers were treated according to the PRF protocol4 with a PC-02 table centrifuge and collection kits provided by Process (Nice, France). Briefly, blood samples were taken without an anticoagulant in 10 ml glass-coated plastic tubes (Becton Dickinson Vacutainer, Franklin Lakes, NJ, USA) and immediately centrifuged at 3000 rpm for 12 minutes. A fibrin clot formed in the middle part of the tube, while the upper part contained acellular plasma, and the bottom part contained red corpuscles (Fig 1a). The fibrin clot was easily separated from the lower part of the centrifuged blood (Fig 1b). The PRF clot was gently pressed into a membrane with sterile dry gauze (Fig 1c).

image

Figure 1.  (a) PRF formed in the middle part of the tube. The upper part contained acellular plasma, and the bottom part contained red corpuscles. (b) The fibrin clot was easily separated from the lower part of the centrifuged blood. (c) The PRF clot was gently pressed between two layers of sterile dry gauze to form a membrane.

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Cell cultures

After approval by the Hospital Review Board, fibroblasts were obtained from the periodontal ligament of premolar teeth from two patients undergoing extraction for orthodontic reasons as described previously.14,15 After extraction, the teeth were placed in Petri dishes containing Dulbecco’s Modification of Eagle’s Medium (DMEM) (GIBCO; Grand Island, NY, USA), 100 units of penicillin and 100 μg of streptomycin/ml. To avoid contamination from the gingiva, the periodontal ligament was carefully removed from the middle third of the root with a scalpel. The fragments were grown in DMEM supplemented with 10% foetal calf serum (FCS) and antibiotics. Cultures were maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. Confluent cell layers were treated with 0.25% trypsin and 0.05% EDTA for five minutes. Aliquots of separated cells were subcultured and then used between the 3rd to 8th passages.

Western blot for p-ERK and OPG

Confluent PDLFs were trypsinized, counted and plated at a concentration of 5 × 104 cells in a 60 mm culture dish and allowed to achieve confluence. Nearly confluent monolayers of cells were washed with serum-free DMEM for 24 hours and immediately thereafter exposed to PRF for the indicated times. Cell lysates were collected on days 1, 3 and 5 as described previously.16 Briefly, cells were solubilized with SDS-solubilization buffer (5 mM EDTA, 1 mM MgCl2, 50 mM Tris-HCl, pH 7.5 and 0.5% Trition X-100, 2 mM phenylmethysulfonyl fluoride and 1 mM N-ethylmaleimide) for 30 minutes on ice. Cell lysates were then centrifuged at 12 000g at 4 °C and the protein concentrations determined with Bradford reagent using bovine serum albumin (BSA) as standards. Equivalent amounts of total protein per sample of cell extracts were run on a 10% SDS-PAGE and immediately transferred to nitrocellulose membranes. The membranes were blocked with PBS containing 3% BSA for 2 hours, rinsed and then incubated with primary antibodies, anti-p-ERK (Cell Signaling Technology, Beverly, MA, USA) (1:1000) or anti-OPG (Abcam, Cambridge, MA, USA) (1:1000) in PBS containing 0.05% Tween 20 for 2 hours. After three washes with Tween 20 for 10 minutes, the membranes were incubated for 1 hour with biotinylated secondary antibody diluted 1:1000 in the same buffer, washed again as described above and treated with 1:1000 streptavidin-peroxidase solution for 30 minutes. After a series of washing steps, the reactions were developed using diaminobenzidine (Zymed, South San Francisco, CA, USA). The intensities of the obtained bands were determined using a densitometer (AlphaImager 2000; Alpha Innotech, San Leandro, CA, USA). Each densitometric value was expressed as the mean ± SD.

Measurement of ALP activity

The cell lysates were sonicated in an ice bath, centrifuged at 1500 g for 5 minutes, and the ALP activity measured in supernatant by ALP assay mixtures containing 0.1 M 2-amino-2-methyl-1-propanol, 1 mM MgCl2 and 8 mM p-nitrophenyl phosphate disodium. After incubation at 37 °C for 30 minutes, the reaction was stopped with 0.1 N NaOH and the absorbance was read at 405 nm. The optical density (OD) of control cells was considered to be 100%. The relative ALP activity of PDLFs was calculated by the formula: (OD of experimental sample/OD of control cells) × 100%. Each value was expressed as the mean ± SD.

Patient population

Six systemically healthy patients (three males and three females) with periodontal infrabony defects were recruited. Inclusion criteria consisted of patients having infrabony defects with a probing depth >5 mm or mandibular grade II furcation defects following initial periodontal therapy. Radiographic evidence of infrabony defects or furcation invasion had to exist. The study protocol was approved by the Institutional Review Board of Chung Shan Medical University Hospital. The surgical procedure and possible alternatives were discussed with the patients.

Application of PRF in periodontal defects

PRF was obtained from each patient according to the protocol as described above. During periodontal surgery, mucoperiosteal flaps were raised and inflammatory periodontal tissues were removed. After this, complete debridement of the infrabony defects, as well as scaling and root planing were achieved (Fig 2a). Minced PRF with bioactive glass (Unigraft; Unicare Biomedical, Laguna Hills, CA, USA) were applied to the defect walls and root surfaces. The PRF/bioactive glass was then tightly packed in the furcation area using amalgam condensers to the vertical level corresponding to the roof of the furcation. In the horizontal direction, the grafting materials were flushed with the bone present on the most apical area of the furcation (Fig 2b). The PRF membrane was adapted over the grafted defect and above the CEJ (Fig 2c). The flaps were repositioned to their presurgical levels and sutured with silk utilizing an interrupted technique (Fig 2d). After the operation, patients were prescribed systemic antibiotics (Cephalosporin 250 mg, qid. 3 days), non-steroidal anti-inflammatory drugs (Cataflam, 25 mg, qid. 3 days) and 0.12% chlorhexidine rinse (twice a day for 4 weeks). Sutures were removed after 7 days.

image

Figure 2.  Clinical application of PRF for case I. (a) Infrabony defects and the grade II furcation invasion of right mandibular first molar. (b) Minced PRF/bioactive glass were applied to the defect walls, root surfaces and tightly packed in the furcation area. (c) PRF membrane was applied to cover the osseous defects. (d) PRF membrane was adapted over the grafted defect and above the CEJ.

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Clinical evaluation

Clinical outcome measures including probing depth, gingival recession and clinical attachment level were recorded at preoperation and six-months postoperation. Periapical radiographs were obtained from the grafted teeth before the operation and 12 months after periodontal regeneration therapy. The radiographs were obtained with a paralleling technique using the Rinn system. All clinical measurements were conducted by the first author. Intra-examiner reproducibility was assessed by duplicate probing depth, gingival recession and clinical attachment level measurements. Intra-examiner intra-class correlation was 95%.

Statistical analysis

Triplicate experiments were performed throughout the in vitro study. All assays were repeated three times to ensure reproducibility. Statistical analysis was by one-way analysis of variance (ANOVA). Differences between treatments were analysed by Duncan’s test.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

PRF was found to increase ERK phosphorylation in PDLFs in a time-dependent manner (p < 0.05) (Fig 3a). The quantitative measurement was made by the AlphaImager 2000 (Fig 3b). Levels of p-ERK activity increased about 2.9, 3.3 and 2.9 fold after exposure of PRF at day 1, 3 and 5, respectively (p < 0.05).

image

Figure 3.  (a) Kinetics of p-ERK expression in PDLFs exposed to PRF for 1, 3 and 5 days, respectively by Western blot analysis. β-actin was performed in order to monitor equal protein loading. (b) Levels of p-ERK protein treated with PRF were measured by AlphaImager 2000. The relative level of p-ERK protein expression was normalized against β-actin signal and the control was set as 1.0. Optical density values represent the mean ± SD. * Represents significant difference from control values with p < 0.05.

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PRF was found to upregulate OPG protein expression in PDLFs during the 5-day culture period (p < 0.05) (Fig 4a). The quantitative measurement by the AlphaImager 2000 is shown in Fig 4b. The levels of the OPG increased about 2.0, 2.6 and 2.7 fold at day 1, 3 and 5, respectively (p < 0.05).

image

Figure 4.  (a) Kinetics of OPG expression in PDLFs exposed to PRF for 1, 3 and 5 days, respectively by Western blot analysis. β-actin was performed in order to monitor equal protein loading. (b) Levels of OPG protein treated with PRF were measured by AlphaImager 2000. The relative level of OPG protein expression was normalized against β-actin signal and the control was set as 1.0. Optical density values represent the mean ± SD. * Represents significant difference from control values with p < 0.05.

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The effects of PRF on the ALP activity were determined by substrate assay. ALP activity was found to be significantly enhanced after PRF treatment (p < 0.05) (Fig 5). The relative ALP activities were 1.4, 1.9 and 1.6 fold after exposure to PRF as compared with the control (p < 0.05).

image

Figure 5.  Kinetics of ALP activity in PDLFs exposed to PRF for 1, 3 and 5 days, respectively by substrate assay. The percentages of ALP activity in the presence of PRF relative to that in the control are shown. The results were averaged from three independent experiments. * Denotes significant differences from control values with p < 0.05.

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Healing of experimental sites was uneventful, and no cases of flap necrosis or infection were observed. All six patients completed the clinical study. Typically, wounds appeared completely healed at 3 weeks postoperatively. Postoperative clinical parameters showed that all grafted sites achieved probing depth reduction and clinical attachment gain (Table 1). Recession was determined from the CEJ to the free gingival margin. The results showed a slight increase of 0.56 mm and 0.94 mm gingival recession at the buccal and lingual side, respectively.

Table 1.   Changes in clinical parameters of grafted teeth
 Preop probing depthPostop 6M probing depthProbing depth reductionAttachment gainGingival recession
  1. Changes in probing depth as measured from the gingival margin and changes in gingival recession and clinical attachment level as measured from the CEJ.

Buccal (B)5.39 ± 1.972.50 ± 0.712.89 ± 1.812.33 ± 1.680.56 ± 0.62
Lingual (L)5.44 ± 2.202.56 ± 0.862.78 ± 1.632.17 ± 1.890.94 ± 0.42
B + L5.42 ± 2.062.53 ± 0.772.83 ± 1.702.25 ± 1.760.75 ± 0.55

PRF application after 12 months follow-up exhibited radiographic evidence that the defect was filled with bone-like dense tissue. Radiographic density clearly increased in infrabony defects and furcation areas (Fig 6). There appeared to be new bone healing in the bony defects.

image

Figure 6.  The periapical radiography of (a) case II and (b) case III after 12 months follow-up period. The images exhibited radiographic defects filled with bone-like dense tissue.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

PRF prepared by Choukroun’s technique is produced naturally without the addition of thrombin, and it is hypothesized that PRF has a natural fibrin framework and can protect growth factors from proteolysis.17 Thus, growth factors can keep their activity for a relatively longer period and stimulate tissue regeneration effectively. Recently, PRF was found to stimulate human gingival fibroblast, osteoblast cell line U2OS and PDLF cell proliferation as a mitogen.11 ERK is known to be involved in cell proliferation. In this study, phosphorylation of ERK was increased by PRF in PDLFs. Similar results were found by Chang et al.18 who found that PRF can upregulate p-ERK expression in human osteoblasts. Thus, the cell proliferation enhanced by PRF may be via p-ERK signal transduction pathway.

OPG has been identified as a soluble member of the tumour necrosis factor superfamily that inhibits osteoclast formation, function and survival by competing with the binding of RANKL.19 In the present study, the expression of OPG was upregulated by PRF. PRF was consistently reported to increase OPG protein expression in human osteoblast cell line18 and pulp cells.20 Taken together, PRF increased the secretion of OPG, suggesting that the enhancement of OPG secretion could inhibit osteolytic activity.

ALP is a membrane-bound glycoprotein, which is one of the osteogenic differentiation markers considered to indicate the presence of osteoblast and the formation of new bone. The expression of ALP has indicated osteoblast-like characteristics in human PDLFs.21 In the present study, ALP activity was elevated by PRF in a time-dependent manner. Similar results have reported that PRF could increase ALP activity in osteoblasts6,22 and human pulp cells20in vitro. These results indicated that PRF may contribute to the differentiation of human PDLFs into osteoblasts.

To the best of our knowledge, this is the first report on the use of PRF for periodontal infrabony defects. In these cases, the reduction in pocket depth was found after PRF application. Pocket depth reduction is not only a desirable outcome of periodontal regenerative procedures, but may also be the most important parameter in patient care for the clinician since it directly impacts one’s ability to instrument a treated area during maintenance appointments. An important clinical outcome of a periodontal regenerative procedure is clinical attachment gain. This gain might have been the result of true periodontal regeneration via new attachment or, alternatively, healing by repair, which implies the presence of a long junctional epithelium between the newly regenerated tissues and the root surface.23

PRF application exhibited the radiographic defects filled in grafted teeth. Radiographic evaluation is a non-invasive examination for bony defects repair. However, bone fill data derived from surgical re-entry are important to substantiate routine postoperative measurement data. In addition, histology of the treated periodontal infrabony defects is the only reliable method to determine the nature of the periodontal soft and hard tissue interface.

PRF is derived from an autogenous preparation of concentrated platelets without any manipulation and is widely used in implant dentistry as a vector for cell growth factors. In this study, PRF enhanced p-ERK, OPG and ALP expression. The application of PRF for periodontal osseous defects achieves probing depth reduction, clinical attachment gain and radiographic defect fills. Thus, PRF as a natural and optimized blood clot seemed the adequate adjuvant to secure this technique and to improve the periodontal infrabony defects.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References
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    Choukroun J, Diss A, Simonpieri A, et al. Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part V: histologic evaluations of PRF effects on bone allograft maturation in sinus lift. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;101:299303.
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    Lacey DL, Timms E, Tan HL, et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 1998;93:165176.
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    Chang YC, Hsieh YS, Lii CK, Huang FM, Tai KW, Chou MY. Induction of c-fos expression by nicotine in human periodontal ligament fibroblasts is related to cellular thiol levels. J Periodontal Res 2003;38:4450.
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    Chang TY, Tsai CH, Chang YC. The upregulation of heat shock protein 47 in human gingival fibroblasts stimulated with cyclosporine A. J Periodontal Res 2010;45:317322.
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    Lundquist R, Dziegiel MH, Agren MS. Bioactivity and stability of endogenous fibrogenic factors in platelet-rich fibrin. Wound Repair Regen 2008;16:356363.
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    Chang IC, Tsai CH, Chang YC. Platelet-rich fibrin modulates the expression of extracellular signal-regulated protein kinase and osteoprotegerin in human osteoblasts. J Biomed Mater Res A 2010;95:327332.
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    Hofbauer LC, Khosla S, Dunstan CR, Lacey DL, Boyle WJ, Riggs BL. The roles of osteoprotegerin and osteroprotegerin ligand in the paracrine regulation of bone resorption. J Bone Miner Res 2000;15:212.
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    Huang FM, Yang SF, Zhao JH, Chang YC. Platelet-rich fibrin increases proliferation and differentiation of human dental pulp cells. J Endod 2010;36:16281632.
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