Platelet-rich Fibrin Membrane Combined with Beta-Tricalcium Phosphate for Treatment of Infrabony Defects in Chronic Periodontitis: A Case Series
Corresponding author: Dr. Kazuhiro Okuda, Division of Periodontology, Department of Oral Biological Science, Niigata University Graduate School of Medical and Dental Sciences, 2-5274 Gakko-cho, Chuo-ku, Niigata 951-8514, Japan, Tel: +81 25 227 2870; Fax: +81 25 227 0808; E-mail: firstname.lastname@example.org
Recently, Platelet-rich fibrin (PRF) was introduced by Choukroun and his co-workers [12,13]. This group of investigators developed a novel technique to clot liquid PRP solely by stimulating the endogenous coagulation pathway. PRF preparations, when compared to PRP preparations, are less influenced by operator skills. This so-called “second generation of PRP” is designated PRF and has been increasingly substituted for PRP in regenerative medicine. In a recently published in vitro study reported by Kobayashi and co-workers, PRF preserved bioactive growth factors such as PDGF-BB, EGF, FGF-4, IGF-II, PDGF-AB and VEGF-D, and stimulated an increase in the number of new blood vessels .
A number of inorganic synthetic graft materials are available for use in the treatment of periodontal infrabony defects. Histological evidence indicated that synthetic grafts act almost exclusively as biological fillers, with scant bone fill and very limited connective tissue regeneration [15, 16]. β-TCP is included in the synthetic absorbable graft materials, has biocompatibility and based on histological studies has demonstrated osteoconductivity [17-19]. On the other hand, β-TCP has not been shown to have the potential for initiating, enhancing or stimulating new attachment apparatus and new blood vessel formation. Therefore, the use of PRF in combination with the osteoconductive, synthetic scaffold β-TCP granules for periodontal regeneration therapy offers an interesting and potentially clinically useful modality to the clinician in treating periodontal osseous defects in humans. Therefore, the purpose of this investigation is to present thirteen cases from a twelve month clinical trial addressing this novel approach of regenerating infrabony osseous defects in humans.
Material and Methods
Patients with advanced chronic periodontitis were recruited and scheduled to receive periodontal therapy at Niigata University Medical and Dental Hospital. The study design and consent form were approved by the ethical committee for human subject use at Niigata University Medical and Dental Hospital in accordance with the Helsinki Declaration of 1975 as revised in 2013. The criteria for inclusion of patients and periodontal sites in the study were 1) non-smoking, free of systemic complications and no history of allergies, 2) no use of antibiotics over the previous 6 months prior to treatment, 3) no treatment for periodontitis during the previous 2 years, 4) presence of one infrabony defect with a probing depth (PD) ≥6 mm, clinical attachment level (CAL) ≥6 mm, osseous defect depth estimated to be ≥3 mm as measured radiographically and 5) presence of at least 2 mm of keratinized gingiva on the facial aspect of the selected tooth.
Prior to the surgical procedures, initial periodontal therapy was performed on all patients. Presurgical treatment consisted of stringent plaque control repeated until patients achieved a Modified O’Leary plaque score  of 10 % or less, full-mouth scaling and root planing under local anesthesia, and occlusal adjustment if trauma from occlusion was present. After completion of the initial therapy, a re-evaluation examination was performed 3 months later to determine patient response to the treatment provided and to confirm the need for periodontal surgery. Fifteen patients were initially enrolled in this study.
Clinical measurements included probing depth (PD), measured as the distance from the free gingival margin to the probeable base of the pocket, clinical attachment level (CAL), measured from the cemento-enamel junction (CEJ) to the base of the pocket by using a calibrated color-coded periodontal probe to the nearest mm (HuFriedy Mfg., Inc, Chicago, IL, USA) and customized acrylic stents with a guiding groove. For radiographic measurements, a commercially available film holder device (Hanshin Technical Laboratory, Ltd., Hyogo, Japan) was modified by placing registration material on the bite block to index the dentition. Standardized radiographs using a paralleling cone technique with positioning aids, were taken at baseline, 3, 6 and 12-month post-surgical evaluation time periods. Radiographic infrabony defect depth (IBD) was assessed using the method described by Cardaropoli and Leonhardt in 2002 . Briefly, the IBD was measured as the radiographic vertical dimension between the projection of the osseous crest adjacent to the root surface (BCP) and the most coronal osseous level adjacent to the root surface where the periodontal ligament space was considered to have a normal width (BoBD). The infrabony osseous defect was then measured by the following calculation: IBD=BCP-BoBD. The twelve-month healing result of each treated osseous defect site was assessed and the difference between baseline and twelve months for the clinical values (PD and CAL) and radiographic values (IBD) were determined.
The PRF membrane preparation procedure was described in a previous report . Briefly, blood was collected from each patient using butterfly needles (21G × 3/4’’; NIPRO, Osaka, Japan) and VacutainerTM tubes (Japan Becton, Dickinson and Company, Tokyo, Japan). To prepare the PRF, the collected blood samples were immediately centrifuged by a Medifuge centrifugation system (Silfradent S. r. l., Santa Sofia, Italy). Preparation of the PRF membrane is shown in Figure 1. After eliminating the red thrombus, the resulting PRF preparation (Figure 1(a)) was compressed by the PRF compression device (Figure 1(b)). The stainless steel PRF compression device developed for PRF membrane preparation is composed of two spoon shaped parts. The clearance of both spoon parts was adjusted to be 1 mm. Thus, when the PRF clot was compressed with this device, a standard 1-mm thick PRF membrane was consistently prepared.
Figure 1. Preparation of the PRF membrane.
a) Platelet-rich fibrin created just after centrifugation
Periodontal surgical procedures were performed on an outpatient basis under aseptic conditions by two trained periodontal clinicians (authors KO and YN). After providing local anesthesia to patients, crevicular incisions were made and full-thickness mucoperiosteal flaps were elevated. Vertical releasing incisions were performed only if necessary for betteraccess or to achieve more favorable closure of the surgical site. The surgical procedure fully exposed the infrabony defects and preserved the marginal gingiva and interdental tissue. Meticulous defect debridement and root planing were carried out to remove visible subgingival plaque, calculus, inflammatory granulation tissue and pocket epithelium. The surgical sites were thoroughly rinsed with sterile saline and care was taken to keep the area free of saliva and blood. The β-TCP granule graft material (Cerasorb®M; curasan AG, Kleinostheim, Germany) was then reconstituted in sterile saline and placed into the defects using amalgam condensers to the vertical height of the corresponding adjacent bone level of the infrabony defect. Then, PRF membranes were overlaid onto the β-TCP granule. The surgical flaps were repositioned to their presurgical levels and sutured with 5-0 nylon suture utilizing an interrupted, vertical mattress technique. Postoperative care included systemic administration of cefcapene pivoxil hydrochloride (FLOMOX ®; Shionogi & Co., Ltd., Osaka, Japan) at 300 mg per day for five days and a 5% povidone iodine (ISODIN®; Meiji Seika Pharma Co., Ltd., Tokyo, Japan) rinse three times daily for six weeks. Sutures were removed at two weeks postsurgery. After suture removal, patient plaque control using the roll tooth brushing technique utilizing an ultra soft toothbrush was resumed at the surgically treated sites. Supragingival professional tooth cleaning was also performed weekly for the first six weeks postsurgery and thereafter the patients were recalled once a month up to twelve months post-surgery for oral hygiene reinforcement and prophylaxis.Statistical analysis
Taking into account the paired nature of the changes from baseline to 12 months, the Wilcoxon signed-rank matched pair test was performed for the pairwise statistical analysis of these data. The null hypothesis was rejected when the risk percentage was below 5% (p <0.05).
Of the 15 patients initially enrolled in thie clinical trial, 13 (7 men and 6 women) completed the study (13 sites). The two patients who were dropped from the data analysis did not return for all their follow-up appointments. Age range of subjects was 40 to 65 years with a mean age of 48.1 ± 6.3 years. There were no patient reported infectious episodes and no other adverse complications associated with treatment over the 12 month time period of this study.
Clinical and radiographic results from 13 patients at baseline and after 3, 6 and 12 months following treatment of infrabony defects with the PRF membrane and β-TCP phosphate are shown in Table 1. Patient compliance with the supportive periodontal therapy was excellent, and the 13 patients included in the data analysis visited the clinic monthly and received maintenance care which included oral hygiene instruction, mechanical tooth polishing and scaling. Patient’s oral hygiene level and infection control were maintained at a high level throughout the study period. Accordingly, the full-mouth plaque score (FMPS) and full-mouth bleeding score (FMBS) remained <10% throughout the entire study, and no significant differences were seen between the evaluation points (data not shown). Individual infrabony defect location and morphology is summarized as follows; Seven defects were located in the maxilla and six were in the mandible. Of the 13 infrabony defects, 3 were two-wall defects and 10 were three-wall defects. The 12-month results after treatment demonstrated the mean PD, CAL and IBD values were improved significantly when compared to baseline (PD: 3.1 ± 0.3 mm at 12-months versus 7.9 ± 1.6 mm at baseline, p <0.01; CAL: 6.6 ± 1.3 mm at 12-months versus 9.5 ± 1.9 mm at baseline, p <0.01; IBD: 1.9 ± 1.0 mm at12-months versus 5.1 ± 1.9 mm at baseline, p <0.01).
* PD = Probing Depth, CAL = Clinical Attachment Level, IBD = Radiographic Infrabony Defect Depth.† Statistical significance level between baseline and 12 months in the treatment group. Statistical significant, p <0.01.
Table 1. P robing depth, clinical attachment level and radiographic infrabony defect depth (mean ± SD) at baseline and after three, six and twelve months following treatment of infrabony defects with the PRF membrane and beta-tricalcium phosphate.
Radiographic and clinical observations including treatment of an infrabony osseous defect is shown in Figures 2 and 3.Case 1A 49-year-old Japanese male, presented with radiographic evidence of bone loss on the mesial surface of the maxillary left canine (Figure 2(a)). The clinical measures of PD and CAL of 9 and 12 mm, respectively, were observed at baseline (Figure 2(b)). At the time of periodontal surgery, a 5-mm-incisal-apical by 5 -mm- mesio-distal osseous defect was observed. A threewalled infrabony osseous defect on the mesio-labial surface of the canine was revealed (Figure 2(c)). After debridement, β-TCP granules were placed into the osseous defect and overlaid with the PRF membrane (Figure 2(d, e)), and then sutured (Figure 2(f)). At 12 months postsurgery, PD and CAL measurements were 3 and 6 mm, respectively (Figure 2(g)). Increased radiopacity was apparent on the treated mesial surface of the canine (Figure 2(h)).
b) Baseline clinical appearance on the mesial surface of the maxillary left canine.
Case 2A 47-year-old Japanese male, presented with radiographic evidence of bone loss on the distal surface of the maxillary right first molar (Figure 3(a)). The clinical measures of PD and CAL of 9 and 9 mm, respectively, were observed at baseline (Figure 3(b)). At the time of periodontal surgery, a 6-mm-incisal-apical by 3 -mm- mesio-distal osseous defect was observed. A two-walled infrabony osseous defect on the distal surface of the first molar was revealed (Figure 3(c)). After debridement, a β-TCP granule graft was placed into the osseous defect and overlaid with the PRF membrane (Figure 3(d, e)), and then sutured (Figure 3(f)). At 12 months postsurgery, PD and CAL measurements were 3 and 6 mm, respectively (Figure 3(g)). Increased radiopacity was apparent on the treated distal surface of the first molar (Figure 3(h)).
Figure 3 (Case 2). Radiographic and clinical observations along with treatment of an infrabony osseous defect using the PRF membrane and β-TCP.
b) Baseline clinical appearance on the distal surface of the maxillary
right first molar.
c) Intraoperative facial view of the 2-walled infrabony defect.
The factors very likely contributing to these more favorable clinical results would be the significant increase in the number of matured blood vessels as well as angiogenic growth factors such as PDGF and VEGF which would provide greater regeneration potential of the graft. In a previous in vitro and animal study evaluating PRF using an ELISA assay, cell culture and scratch assay, western blotting analysis, the chorioallantoic membarane (CAM) assay and histological and immunohistochemical examination, it was found that 1) angiogenic growth factors such as PDGF and VEGF were concentrated when compared with platelet-poor plasma, 2) PRF preparations had an enhanced effect on phosphorylation of VEGFR2 in human umbilical vein endothelial cells (HUVECs) and 3) PRF preparations favorably improved new blood capillary formation, and 4) also favorably impacted the thickness and structure of the CAM and the formation of mature blood vessels in the CAM .Another factor that should be considered in interpreting our favorable clinical results in treating human osseous defects is the network of fibrin fibers which is one of the major structural components in PRF. Previously it has been observed by SEM examination that platelets aggregated and attached on the fibrin surface of PRF membranes [14, 25], and therefore, the PRF membrane may function to serve as a storage of growth factors before their release from platelets. In addition, the PRF membrane with its fibrin network may act like a bandage that accelerates the healing of wound edges. The PRF membrane may also provide significant postoperative protection of the surgical site and may hasten the integration and remodeling of the grafted biomaterial [24, 25]. Among the factors likely contributed by the PRF membrane to the favorable clinical results would be the positive clinical effect of β-TCP granules serving as a scaffold to maintain space making and the osteoconductivity nature of the β-TCP grafting material [22-24].
Within the clinical trial design limits of this case series, PRF membranes in combination with β-TCP granules demonstrated a favorable clinical improvement in treating infrabony osseous defects. In the future, clinical studies involving a greater number of human subjects in a randomized controlled clinical trial study design and monitoring for a longer period of time would be necessary to definitively prove the clinically favorable results we reported here for this new tissue engineered grafting procedure in treating periodontal osseous defects.
Declaration of interests:The authors have no commercial, proprietary, or financial interest in the products or companies described in this article.
This study was supported by the Japanese Society for the Promotion of Science (KAKENHI; Grant No.24390465
2.Camelo M, Nevins ML, Schenk RK, Lynch SE, Nevins M. Periodontal regeneration in human class II furcations using purified recombinant human platelet-derived growth factor BB (rhPDGF-BB) with bone allograft. Int J Periodontics Restorative Dent. 2003, 23(3):213-225.
3.Okuda K, Momose M, Murata M, Saito Y, Inoie M et al. Treatment of chronic desquamative gingivitis using tissue engineered human cultured gingival epithelial sheets: a case report. Int J Periodontics Restorative Dent. 2004, 24(2):119-125.
4.Nevins M, Giannobile EV, McGuire MK, Kao RT, Mellonig JT et al. Platelet-derived growth factor stimulates bone fill and rate of attachment level gain: Results of a large multicenter randomized controlled trial. J Periodontol. 2005, 76(12): 2205-2215.
5.Yamamiya K, Okuda K, Kawase T, Hata K, Wolff LF et al. Tissue-engineered cultured periosteum used with platelet- rich plasma and hydroxyapatite in treating human osseous defects. J Periodontol. 2008, 79(5):811-818.
8.Okuda K, Nakajima Y, Irie K, Sugimoto M, Kabasawa Y et al. Transforming growth factor-β1 coated β-tricalcium phosphate pellets stimulating healing of experimental bone defects of rat calvariae. Oral Dis. 1995, 1(2): 92-97.
9.Okuda K, Murata M, Sugimoto M, Saito Y, Kabasawa Y et al. TGF-β1 influences early gingival wound healing in rats: An immunohistochemical evaluation of stromal remodeling by extracellular matrix molecules and PCNA. J Oral Pathol Med. 1998, 27(10): 634-669.
10.Okuda K, Kawase T, Momose M, Murata M, Saito Y et al.Platelet-rich plasma contains high levels of platelet-derived growth factor and transforming growth factor-β and modulates the proliferation of periodontally related cells in vitro. J Periodontol. 2003, 74(6):849-857.
11.Okuda K, Tai H, Tanabe K, Suzuki H, Saito Y et al. Platelet- rich plasma combined with a porous hydroxyapatite graft for the treatment of intrabony periodontal defects in humans: a comparative controlled clinical study. J Periodontol. 2005, 76(6): 890-898.
12.Jaaskelainen SK. Pathophysiology of primary burning mouth syndrome. Clin Neurophysiol. 2012, 123(1): 71-77Dohan DM, Choukroum J, Diss A, Dohan SL, Dohan AJ et al. Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part I: technological concepts and evolution. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006, 101(3): e37-e44.
13.KossDohan DM, Choukroum J, Diss A, Dohan SL, Dohan AJ et al. Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part II: platelet-related biologic features. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006, 101(3): e45-50.
16.Stahl SS, Froum S. Histologic evaluation of human intraosseous healing responses to the placement of tricalcium phosphate ceramic implants in human periodontal defects. Three to eight months. J Periodontol 1986, 57(4): 211-217.
17.Hori T, Makabe K, Nemoto K, Asada T. Hypersalivation induced by olanzapine with fluvoxamine. Prog Neuropsychopharmacol Biol Psychiatry. 2006, 30(4):758-760.
18.Takabatake K, Yamachika E, Tsujigiwa H, Takeda Y, Kimura M et al. Effect of geometry and microstructure of honeycomb TCP scaffolds on bone regeneration. J Biomed Mater Res A. 2014, 102(9): 2952-2960.
19.Hainich J, von Rechenberg B, Jakubietz RG, Jakubietz MG, Giovanoli P et al. Osteoconductive behavior of beta-tricalcium phosphate ceramics in osteoporotic, meyaphyseal bone defects of the distal radius. Handchir Mikrochir Plas Chir. 2014, 46(1):12-17.
23.Pradeep AR, Sharma A. Treatment of 3-wall intrabonydefects in chronic periodontitis subjects with autologous platelet rich fibrin-a randomized controlled clinical trial. J Periodontol. 2011, 82(12):1705-1712.
25.Kawase T. Platelet-rich plasama and its derivatives as promising bioactive materials for regenerative medicine: basic principles and concepts underlying recent advances. Odontology. 2015, 103(2): 126-135.