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Article Type: Randomized Clinical Trial
Human intrabony defect regeneration with rhFGF-2 and hyaluronic acid – a randomized controlled clinical trial Ronaldo B. SANTANA, DDS, MScD, DSc †, Carolina Miller Mattos de SANTANA, DDS, MScD, DScD † † Department of Periodontology, Universidade Federal Fluminense. Dental School. Niteroi, Rio de Janeiro, Brazil.
Short Title: Periodontal regeneration with rhFGF-2
KEY WORDS: periodontitis, periodontal regeneration, rhFGF-2.
Summary: rhFGF-2 is superior to open flap debridement in human intrabony defects
Corresponding Address: Ronaldo B Santana, DDS, MScD, DSc Universidade Federal Fluminense Department of Periodontology Rua São Paulo 28 – Praca do Valonguinho Niterói – Rio de Janeiro – Brazil 24000000 E-mail: [email protected] ABSTRACT AIM: The goal of the present study was to evaluate if a biologic hydrogel of recombinant human Fibroblast Growth Factor type 2 (rhFGF-2) in a hyaluronic acid (HA) carrier applied in periodontal intrabony defects would enhance the clinical This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/jcpe.12406 This article is protected by copyright. All rights reserved.
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parameters of regeneration of the periodontal attachment apparatus. MATERIALS AND METHODS: Thirty adult patients were evaluated. Two intra-bony defects present in contralateral quadrants in each patient were randomly allocated for each of the treatment methods employed. Control group (n=30) were treated by open debridement with the papilla preservation flaps, while the test group (n=30) also received a topical application of rhFGF-2/HA in the intrabony defect. The parameters evaluated, at baseline and after one year, were, were probing depth (PD), gingival recession (REC), probing attachment level (PAL) and probing bone level (PBL). The primary outcome measures was PAL gain. RESULTS: Test sites exhibited significantly more PD reduction (5.5 versus 2.9mm), PAL gains (4.8 vs 2.2mm)
and shallower residual PD (4.2 versus 6.6mm) than
controls. Moreover, residual PD smaller than 5mm (100 versus 0%) and PAL gain > 4mm (60 versus 20%) was significantly more frequent in the test group. CONCLUSION: Application of rhFGF-2/HA significantly improved clinical parameters of periodontal wound healing one year after treatment.
Clinical Relevance Scientific rationale for the study: rhFGF-2 is being evaluated as a candidate therapy for periodontal/bone regeneration. The present study reports on the use of rhFGF-2 in a Hyaluronic Acid (HA) gel carrier in periodontal regeneration in humans. Principal findings: Application of rhFGF-2/HA enhances the clinical parameters of periodontal regeneration.
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Practical implications: rhFGF-2/HA may be clinical useful for regenerative periodontal procedures.
Source of Funding: The study was self-funded by the authors and their institution, but Orquest, Inc (Mountain View, CA, USA) provided free materials to be used in the study
Introduction: Regeneration of the lost attachment apparatus is the ultimate goal of periodontal therapy, in conjunction with shallower probing depths (PD), thus, facilitating periodontal maintenance (Santana et al 2009). Despite the presence of periodontal disease, the periodontal attachment apparatus harbours cells with regenerative potential (Schwartz et al 2000; Wennstrom & Lindhe 2002), thus, selective population of the periodontal wound by these cells is one of the main goals of regenerative periodontal therapy. New attachment occurs with the formation of new cementum with inserting collagen fibres over a previously contaminated root surface, while regeneration of the periodontal attachment apparatus also includes the formation of new alveolar bone. Several procedures have been suggested for the regenerative therapy of periodontal lesions, including use of autografts, allografts and xenografts (Sculean et al 2008), Guided Tissue Regeneration (Santana et al 2009; Cortellini et al 2000) and several methods for conditioning of the root (Mariotti 2003, Esposito et al 2009). Despite their partially successful results, these procedures have limited clinical applicability due to unpredictability of results and their dependence on the intrinsic healing potential of the host. Use of bioactive substances of-
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ported in the periodontal literature for the treatment of similar periodontal defects with other regenerative therapies, including membranes, bone grafts, enamel matrix proteins or growth factors such as rhPDGF or rhGDF (Trombelli et al. 2002, Mariotti 2003, Needleman et al. 2005, Esposito et al 2009, Graziani et al 2012, Susin & Wikesko 2013, Lee & Wikesjo 2014). Two previous studies have evaluated the effects of rhFGF-2 in a hydroxypropyl cellulose gel formulation (Kitamura et al 2008; Kitamura et al 2011) and reported a relatively modest increase in radiographic bone level (1.85 ± 1.75 mm) and PAL gain (2.18 ± 1.33 mm) 9 months after treatment of comparatively deeper intrabony defects. These data apparently suggest an enhanced regenerative potential of the rhFGF-2/HA hydrogel reported in the present study. Further studies evaluating different carriers for rhFGF-2 and growth factor dosing may further clarify the relative regenerative potential of new formulations. A limitation of the present study was the fact that no positive control group treated with the HA carrier alone was employed. However, the scarce literature available strongly suggests that HA may possess little, if any, regenerative effect when employed alone in surgical therapy (Hunt et al 2001). Engström et al (2001) reported 0.5mm increase in bone height when HA was employed in conjunction with barrier membranes. Ballini et al (2009) reported PAL gains of 2.6mm measured 9 months after treatment with the use of HA in conjunction with autogenous bone grafting in a case series with nine patients. Considering that similar, or even better, results have been documented with the use of autogenous bone grafting alone in the treatment of periodontal defects (Hiatt & Schallhorn 1973; Froum et al 1976; Abolfazli et al 2008), potential positive effects of HA
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2 has been demonstrated in several pre-clinical animal studies (Oi et al 2009; Murakami et al 1999, 2003; Takayama et al 2001; Rossa et al 2003) with accelerated regeneration of cementum and functional periodontal ligament (Sato et al 2004; Nakahara et al 2004, Yang et al 2004). Local application of FGF-2 significantly enhances vascularization and osteogenesis, defect filling and cementum formation (Rossa et al 2003, Sato et al 2004, Nakahara et al 2004).
Therefore, FGF-2 is a potential candidate as a therapeutic molecule due to its anabolic effects on cementum, periodontal ligament and bone and may offer an exciting new alternative in periodontal regenerative surgery. The objective of this study was to evaluate if rhFGF-2 delivered into periodontal defects in a hyaluronic acid (HA) carrier would enhance the clinical parameters of regeneration of the periodontal attachment apparatus. The hypothesis under investigation was that the local application of rhFGF2/HA would significantly enhance periodontal attachment and bone gain in intrabony defects in humans.
Materials and Methods Study Population and Experimental Design The study was designed as a randomized, prospective, split-mouth, controlled clinical trial. It was conducted in accordance with the guidelines of the World Medical Association Declaration of Helsinki (version VI, 2002), after approval of the study design and consent by the Faculty of Medicine, Ethical Committee of Medical Research, Federal Fluminense University (Approval protocol CEP-HUAM/CCM 21/03). Written informed
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consent was obtained from all subjects. Systemically healthy patients with moderate to advanced chronic periodontitis referred to the Department of Periodontology, Faculty of Dentistry, Federal Fluminense University for periodontal treatment of advanced periodontitis were recruited for and treated in the study. Inclusion criteria of the study were: (a) adult subjects with chronic periodontitis presenting (b) radiographic evidence alveolar bone loss at the proximal aspect of the tooth, (c) intrabony defect deeper than 4mm, (d) probing pocket depth >6 mm at the site, (e) unremarkable general health according to medical history and clinical judgment, (f) no medications taken for at least six months and no antibiotics taken for twelve months before the beginning of the study. Subjects who have quit the habit of smoking for at least one year before the beginning of the study were considered as non-smokers and, if the other included criteria were met, were included in the present sample. Exclusion criteria for the study were: (a) subjects with “early onset” or aggressive forms of periodontitis, (b) current smokers, (c) presence of significant systemic diseases (i.e., cancer, AIDS, diabetes), (d) clinical evidence of furcation defects, (e) presence of apical radioleucency and (f) previous lack of cooperation with the maintenance program. Two defects were selected from each patient, and were randomly assigned, immediately before each surgical appointment, by computer generated random sequence allocation (custom randomisation software, Department of Biostatistics, Federal Fluminense University) to one of the two treatment modalities employed: (a) Control: open instrumentation of the root surfaces and bone defects via flap modified papilla preservation flap; (b) Test: open instrumentation of the root surfaces and bone defects via flap modified papilla preservation flap and application of a rhFGF-2/HA in the intrabony defect.
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Treatment allocation was registered in an allocation table sheet that was unavailable to the clinical examiner throughout the study. Both the control and test sites were treated at the same surgical appointment and no information on treatment allocation was provided to the patient. All surgical procedures were performed by a single operator (RBS), which became aware of the site allocation only after completed root planning and conditioning, and immediately before the application of the growth factor in the test sites. A strict surgical protocol emphasizing careful hard- and soft-tissue management, root debridement, wound stability and infection control was employed (Santana et al 2009; Miranda et al 2013). Nonsurgical infection control was performed in the context of cause- related periodontal therapy and consisted of oral hygiene instructions, scaling and root planing, tooth polishing and plaque control measures performed six months prior to regenerative surgery. Patients were re-evaluated three and six months after the completion of the nonsurgical cause-related periodontal therapy and enrolled following the later evaluation appointment.
Clinical Recordings Clinical parameters were assessed using the cemento-enamel junction (CEJ) or, when applicable, another defined landmark, as a fixed reference point. All measurements were recorded using an UNC #15 periodontal probe (PCP-UNC 15, Hu-Friedy, Chicago, IL, USA) by a blinded, trained and calibrated examiner (CMMS), unaware of the treatment provided at baseline and one year after treatment. Measurements were recorded to the nearest mm. Full mouth plaque score (FMPS) was recorded dichotomously as the percentage of total surfaces (four sites per tooth) (O’Leary et al. 1972). BOP was also
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Figure 2 – Representative bone defect treated by control therapy.
A- Baseline clinical view; B - Surgical view after flap elevation; C - Closure (buccal view); D- Clinical view one year after treatment; E- Baseline radiographic aspect; F -Radiographic result one year after treatment
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Flap Design Intracrevicular incisions were made and buccal and lingual mucoperiosteal flaps, including at least one tooth mesial and another distal the tooth being treated, were elevated by blunt dissection. Extreme care was taken to preserve the marginal gingiva and inter-dental tissue to achieve better closure of the treated sites. Thus, the defect-associated inter-dental papilla was accessed with papilla preservation flap techniques (Takey 1985, Cortellini et al. 1995, Cortellini et al. 1999) according to indications. A vertical releasing incision was performed at the mesial aspect of the flap whenever necessary to obtain optimized, tensionless, access to the surgical area.
Root and bone defect debridement The bone defect was meticulously debrided with surgical curettes and ultrasonic instruments with special effort to remove all the inflammatory granulation tissue, but with no intention to perforate the cortical bone walls. Thorough root planing was performed with hand (Hu-Friedy, IL, USA), rotary (Intensiv, Grancia, Switzerland) and ultrasonic instruments (Cavitron, Dentisply, USA), to remove subgingival plaque and calculus until all root surfaces attained a hard glassy surface. During the instrumentation, the flaps were slightly elevated, carefully protected with periosteal elevators, and frequently irrigated with saline.
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Root conditioning and rhFGF-2 application After instrumentation, the root surfaces were washed with saline solution to remove any remaining detached fragments from the defect and surgical field. Then, a freshly prepared tetracycline hydrochloride paste in saline was applied with gentle circular movements on the instrumented and dried root surfaces of both test and control groups for three minutes. After that, the defect area was carefully rinsed with saline. Finally, on the experimental sites, 0.2ml of a gel containing 4mg/ml rhFGF-2 in sodium hyaluronate MW 1.3 x 106 (Ossigel®, Orquest, CA, USA) was applied on the dried root surfaces and bone defect (Figure 1C). The flaps were then repositioned and tension-free primary flap closure was obtained using internal mattress, and single interrupted sutures (5.0 Ethicon Sutures, Johnson & Johnson, NJ, USA or Gore-tex Sutures, W.L Gore, AZ, USA). Representative clinical cases are presented in figures 1 and 2.
Post-Operative Treatment The patients were prescribed systemic antibiotic therapy consisting of 200mg of doxycycline***** the day before surgery, followed by 100 mg daily continued until the 20th day after surgery. No surgical dressing was used. The patients were instructed to continue their regular home hygiene care, except in the operated area, which was cleaned by means of gentle topical applications of 0.2% chlorhexidine gluconate (Perioxidin®, Lacer, Barcelona, Spain) in saturated cotton swabs. Patients were instructed not to brush or floss the surgical area for 4 weeks, when mechanical plaque control was re-instituted. The sutures were removed two weeks after surgery. Hyper-mobile teeth were splinted either before or immediately after the surgical procedure.
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aintenance Schedule Patients were enrolled in a stringent post-operative supportive care programme following surgery (Santana et al 2009). All patients were seen weekly during the first three months and bi-weekly for the next three months. Thereafter, the patients were seen once monthly for the last six-month period of the study. After 1 year, the patients were recalled and re-instructed on proper oral hygiene. Sites were rescaled when indicated.
Radiographic assessments Radiographs were taken with a parallel, standardized, technique at baseline (Eickholz et al. 2004). Radiographs were digitized and analysed using NIH Image Analysis software. The anatomical landmarks for the study included cementum-enamel junction (CEJ), alveolar crest (AC) and base of the defect (BD) (Schei et al. 1959). Radiographic defect depth was measured along the root surface, parallel to the root axis, as the distance between CEJ and BD.
Statistical Analyses Quantitative data were recorded as mean and standard deviations. No data points were missing. Sample size was determined by Power analysis, assuming a standard deviation of the difference of 0.5, which indicated that with a sample of 20 subjects, the study would have >90% power to detect a 1-mm difference in the primary outcome measure PAL gain between the two groups. In order to verify the normality of the data, the kurtosis and skewness curves were used. Because all data were considered to be normal for the parameters analysed, Student’s paired t-test was used for intra-group (baseline versus 12
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months) and inter-group
(test versus control) comparisons. The level of significance
set at 5% was used in all statistical comparisons performed in the software ProStat (Poly Software International, Pearl River, NY, USA) .
Results Thirty adult patients (11 male, 19 female), 39 to 66 year-old (mean age of 49.2 years), meeting the inclusion criteria were treated. Complete data from 60 intrabony defects distributed in 15 incisors (8 test and 7 controls), 13 canines (six test and 7 controls), 18 premolars (9 in each group) and 14 lower molars (seven in each group) were available for statistical comparisons. Post-hoc power calculations assuming µ1equal to 4.4, µ2 equal to 2.2, sigma (σ) equal to 0.4, an alpha value of 0.05 and a sample size (n) of 30, revealed that the study had 100% power to detect the differences in the primary outcome measure. Healing was uneventful for all treated cases in both patient groups. Soft tissues healed within normal limits and no significant visual differences were noted between the treatment groups. No event of malaise, fever or pain was reported or detected. No significant edema, erythema, ulceration or hematoma was noted. Patients maintained an adequate level of plaque control throughout the duration of the study, as evidenced by the FMPS (17.6±2.1) and BOP (19.3±2.3) measurements obtained one year after treatment. Data from baseline clinical (PD, REC, PAL, CEJ-BD), radiographic (JCE-BDX, JCECOX, INTRAX) and trans-surgical (BD, BC, INTRA) measurements from test and control teeth are presented in Table 1. Inter-group measurements did not demonstrate significant differences between the groups, demonstrating homogeneity of the patient popula-
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tion under study. Measurements of distance CEJ-BD obtained after elevation of a mucoperiosteal flap were not significantly different to those obtained by trans-gingival probing demonstrating the equivalence of either method do estimate bone crest relation to the CEJ. Data from clinical measurements (PD, REC, PAL, CEJ-BD) performed in teeth from Experimental and Control groups one year after surgery are presented in table 2. Intra-group evaluations demonstrated reductions in PD, PAL e CEJ-BD, as well as increased REC, demonstrating significant differences between results from baseline and one year after treatment. These results demonstrate that both therapies were effective in significantly reducing periodontal probing depths and bone defects and enhancing probing attachment levels. However, both groups resulted in significant increase in gingival recession at treated sites. Inter-group comparisons demonstrated that the magnitude of changes in PD, PAL and CEJ-BD were significantly bigger in the test group (table 2), suggesting a higher efficacy of the test treatment in enhancing the clinical parameters of wound healing evaluated in the present experimental patient cohort. A comparison of frequency distributions of post-treatment PAL gain and residual PD is presented in table 3. Data demonstrates that the majority (60%) of sites in the test group exhibited a significant amount (>4mm) of PAL gain while in the control group the majority (80%) of sites presented less than 2mm of attachment gain. Moreover, the test group also presented with all sites with less than 5mm of residual PD, with most sites (56%) with residual probing depths shallower than or equal to 4mm. In contrast, all sites in the control group presented with residual PD equal to or deeper than 5mm.
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Discussion This longitudinal, randomized, controlled clinical trial demonstrated that an innovative combination of bioactive substances composed of rhFGF-2/HA applied in periodontal intrabony defects results in significantly more PD reduction, PAL and bone gains, higher frequency of sites with shallower PD and 4mm or more PAL gains than the control group one year after treatment, suggesting that this new treatment modality based on the principles of tissue engineering may demonstrate enhanced clinical outcomes in comparison with the conventional treatment. To the best of our knowledge, this is the first report on the use of a composite rhFGF-2/HA hydrogel in periodontal regeneration. Several systematic reviews with meta-analysis have demonstrated that open flap debridement has been widely employed as the control group in the majority of the studies evaluating regenerative techniques in intrabony defects (Trombelli et al. 2002, Needleman et al. 2005, Esposito et al. 2009, Graziani et al 2012), demonstrating greater variability of the results obtained (Tu et al 2008), probably due to type of surgical technique, flap design, incision and suturing employed (Graziani et al 2012). In line with the results of previous studies in the literature, the magnitude of change in the clinical parameters evaluated were relatively modest in the control group and inferior to the regenerative technique evaluated in parallel (Trombelli et al. 2002, Needleman et al. 2005, Esposito et al. 2009, Graziani et al 2012). The experimental therapy resulted in significantly more PD reduction, PAL gain and bone gains than the traditional open flap instrumentation, clearly demonstrating the bioactivity of the material, and enhancement of the healing in the human intrabony defect environment. The magnitude of changes compares positively with previous results re-
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ported in the periodontal literature for the treatment of similar periodontal defects with other regenerative therapies, including membranes, bone grafts, enamel matrix proteins or growth factors such as rhPDGF or rhGDF (Trombelli et al. 2002, Mariotti 2003, Needleman et al. 2005, Esposito et al 2009, Graziani et al 2012, Susin & Wikesko 2013, Lee & Wikesjo 2014). Two previous studies have evaluated the effects of rhFGF-2 in a hydroxypropyl cellulose gel formulation (Kitamura et al 2008; Kitamura et al 2011) and reported a relatively modest increase in radiographic bone level (1.85 ± 1.75 mm) and PAL gain (2.18 ± 1.33 mm) 9 months after treatment of comparatively deeper intrabony defects. These data apparently suggest an enhanced regenerative potential of the rhFGF-2/HA hydrogel reported in the present study. Further studies evaluating different carriers for rhFGF-2 and growth factor dosing may further clarify the relative regenerative potential of new formulations. A limitation of the present study was the fact that no positive control group treated with the HA carrier alone was employed. However, the scarce literature available strongly suggests that HA may possess little, if any, regenerative effect when employed alone in surgical therapy (Hunt et al 2001). Engström et al (2001) reported 0.5mm increase in bone height when HA was employed in conjunction with barrier membranes. Ballini et al (2009) reported PAL gains of 2.6mm measured 9 months after treatment with the use of HA in conjunction with autogenous bone grafting in a case series with nine patients. Considering that similar, or even better, results have been documented with the use of autogenous bone grafting alone in the treatment of periodontal defects (Hiatt & Schallhorn 1973; Froum et al 1976; Abolfazli et al 2008), potential positive effects of HA
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alone could not be ascertained. It is also important to realize that the magnitude of the difference between the experimental groups of the present study, in favor of the test therapy, for PAL (2.6mm) and bone gains (2.3mm) were 4 to 5-fold higher than in the previous studies. Indeed, the PAL gains reported by Ballini et al (2009) for HA plus autogenous bone was very similar than the amount of PAL gain observed in the control group of the present study. Thus, taken together, these data suggest that the improved results observed in the present study may be attributed to the biological activity of FGF-2, and that HA, although acting as a potentially adequate carrier, apparently did not significantly influence the treatment outcomes. Future controlled clinical studies employing HA alone may be necessary to further clarify this issue. In the present report we used HA as the carrier for rhFGF-2 due to the wellestablished bi-directional biologic relationships between both substances. The binding affinity of FGF-2 to glycosaminoglycans (GAG) with resultant protection from exogenous protease digestion is well characterized (Coltrini et al 1993). On the other hand, FGF-2 positively regulates GAG biosynthesis, especially in the periodontal environment (Shimabukuro et al 2005, 2008). FGF-2 regulates the hyaluronan metabolism in PDL cells by significantly increasing HA production, specifically enhancing mRNA expression of the enzymes hyaluronan synthase 1 and 2 (Shimabukuro et al 2005) and enhancement of the production of membrane-bound heparan sulfate proteoglycan syndecan (Shimabukuro et al 2008). Therefore, FGF-2 and HA share important biological interactions and may present synergistic activities. Previous data suggest little possibility that FGF-2 will cause systemic adverse effects after topical application, since no FGF-2 entered the circulation, and that locally-
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applied FGF-2 was not associated with antibody production (Kitamura et al 2008; Kitamura et al 2011). In accordance, the present population did not demonstrate any significant local or systemic alterations following application of FGF-2, as documented locally by soft tissues healing within normal limits and no significant visual differences between the treatment groups (no significant edema, erythema, ulceration or hematoma). Systemically, no event of malaise, fever or pain was reported or detected. These preliminary findings demonstrated a positive clinical response of human periodontal intrabony defects treated with a regenerative hydrogel composed of rhFGF-2 and HA, in accordance with the principles of tissue engineering, and suggest that this approach may offer potential benefits for the treatment of such lesions. Further studies with other potential combined therapies are warranted to further evaluate the efficacy of this new regenerative material and may further improve the clinical benefits of rhFGF-2/HA in periodontal surgical therapy.
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Acknowledgements: The authors thank Dr James W. Poser, PhD and Orquest, Inc (Mountain View, CA, USA) for the generous gift of the rhFGF-2/HA hydrogel employed in the present study.
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Table 1 – Measurements obtained at baseline MEASUREMENT
CONTROL
TEST
DIFFERENCE
P
PD
9.5±1.5
9.7±1.9
0.2±0.1
0.25 NS
REC
0.9±0.7
0.8±0.6
0.1±0.1
0.28 NS
PAL
10.3±1.3
10.4±1.6
0.1±1.6
0.33 NS
CEJ-BD
11.6±1.3
11.8±1.2
0.2±0.1
0.30 NS
CEJ-BDX
11.2±1.1
11.5±1.3
0.3±0.1
0.13 NS
CEJ-COX
6.1±1.5
6.2±1.9
0.1±1.7
0.42 NS
INTRAX
5.1±1.3
5.3±1.2
0.2±1.2
0.53 NS
BD
12.2±1.5
12.5±1.0
0.3±0.7
0.67 NS
BC
6.9±1.2
6.8±1.6
0.2±1.3
0.65 NS
INTRA
5.3±1.3
5.7±1.2
0.4±1.3
0.28 NS
One Wall
0.7±1.2
0.9±1.1
0.2±1.1
0.77 NS
Two Wall
1.2±1.3
1.4±1.2
0.2±1.3
0.68 NS
Three Wall
3.3±1.0
3.4±1.1
0.3±1.0
0.57 NS
PD = Probing depth, REC = gingival recession, PAL = probing attachment level, CEJ-BD = probing distance between cement-enamel junction and base of bone defect; CEJ-COX = radiographic distance between the CEJ and bone crest; JCE-BDX = radiographic distance between CEJ and the base of the bone defect); INTRAX = radiographic distance between the base of the bone defect and the bone crest; BD = intra-surgical distance between cement-enamel junction and base of bone defect; BC = intra-surgical distance between the CEJ and bone crest; INTRA = intrasurgical distance between the base of the bone defect and the bone crest. Three, Two and One Wall = the depth of the 3-, 2-, and 1-wall sub-components of the bony defect in mm. Data presented as mean ± standard deviation. P = statistical probability. NS = non-significant.
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Table 2 – Inter- and intra-group measurements obtained one year after treatment. MEASUREMENT
CONTROL
TEST
DIFERENCE
P
PDi
9.5±1.5
9.7±1.9
0.2±0.1
0.25 NS
PD1
6.6±1.3
4.2±0.8
2.4±0.1
Article Type: Randomized Clinical Trial
Human intrabony defect regeneration with rhFGF-2 and hyaluronic acid – a randomized controlled clinical trial Ronaldo B. SANTANA, DDS, MScD, DSc †, Carolina Miller Mattos de SANTANA, DDS, MScD, DScD † † Department of Periodontology, Universidade Federal Fluminense. Dental School. Niteroi, Rio de Janeiro, Brazil.
Short Title: Periodontal regeneration with rhFGF-2
KEY WORDS: periodontitis, periodontal regeneration, rhFGF-2.
Summary: rhFGF-2 is superior to open flap debridement in human intrabony defects
Corresponding Address: Ronaldo B Santana, DDS, MScD, DSc Universidade Federal Fluminense Department of Periodontology Rua São Paulo 28 – Praca do Valonguinho Niterói – Rio de Janeiro – Brazil 24000000 E-mail: [email protected] ABSTRACT AIM: The goal of the present study was to evaluate if a biologic hydrogel of recombinant human Fibroblast Growth Factor type 2 (rhFGF-2) in a hyaluronic acid (HA) carrier applied in periodontal intrabony defects would enhance the clinical This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/jcpe.12406 This article is protected by copyright. All rights reserved.
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parameters of regeneration of the periodontal attachment apparatus. MATERIALS AND METHODS: Thirty adult patients were evaluated. Two intra-bony defects present in contralateral quadrants in each patient were randomly allocated for each of the treatment methods employed. Control group (n=30) were treated by open debridement with the papilla preservation flaps, while the test group (n=30) also received a topical application of rhFGF-2/HA in the intrabony defect. The parameters evaluated, at baseline and after one year, were, were probing depth (PD), gingival recession (REC), probing attachment level (PAL) and probing bone level (PBL). The primary outcome measures was PAL gain. RESULTS: Test sites exhibited significantly more PD reduction (5.5 versus 2.9mm), PAL gains (4.8 vs 2.2mm)
and shallower residual PD (4.2 versus 6.6mm) than
controls. Moreover, residual PD smaller than 5mm (100 versus 0%) and PAL gain > 4mm (60 versus 20%) was significantly more frequent in the test group. CONCLUSION: Application of rhFGF-2/HA significantly improved clinical parameters of periodontal wound healing one year after treatment.
Clinical Relevance Scientific rationale for the study: rhFGF-2 is being evaluated as a candidate therapy for periodontal/bone regeneration. The present study reports on the use of rhFGF-2 in a Hyaluronic Acid (HA) gel carrier in periodontal regeneration in humans. Principal findings: Application of rhFGF-2/HA enhances the clinical parameters of periodontal regeneration.
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Practical implications: rhFGF-2/HA may be clinical useful for regenerative periodontal procedures.
Source of Funding: The study was self-funded by the authors and their institution, but Orquest, Inc (Mountain View, CA, USA) provided free materials to be used in the study
Introduction: Regeneration of the lost attachment apparatus is the ultimate goal of periodontal therapy, in conjunction with shallower probing depths (PD), thus, facilitating periodontal maintenance (Santana et al 2009). Despite the presence of periodontal disease, the periodontal attachment apparatus harbours cells with regenerative potential (Schwartz et al 2000; Wennstrom & Lindhe 2002), thus, selective population of the periodontal wound by these cells is one of the main goals of regenerative periodontal therapy. New attachment occurs with the formation of new cementum with inserting collagen fibres over a previously contaminated root surface, while regeneration of the periodontal attachment apparatus also includes the formation of new alveolar bone. Several procedures have been suggested for the regenerative therapy of periodontal lesions, including use of autografts, allografts and xenografts (Sculean et al 2008), Guided Tissue Regeneration (Santana et al 2009; Cortellini et al 2000) and several methods for conditioning of the root (Mariotti 2003, Esposito et al 2009). Despite their partially successful results, these procedures have limited clinical applicability due to unpredictability of results and their dependence on the intrinsic healing potential of the host. Use of bioactive substances of-
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ported in the periodontal literature for the treatment of similar periodontal defects with other regenerative therapies, including membranes, bone grafts, enamel matrix proteins or growth factors such as rhPDGF or rhGDF (Trombelli et al. 2002, Mariotti 2003, Needleman et al. 2005, Esposito et al 2009, Graziani et al 2012, Susin & Wikesko 2013, Lee & Wikesjo 2014). Two previous studies have evaluated the effects of rhFGF-2 in a hydroxypropyl cellulose gel formulation (Kitamura et al 2008; Kitamura et al 2011) and reported a relatively modest increase in radiographic bone level (1.85 ± 1.75 mm) and PAL gain (2.18 ± 1.33 mm) 9 months after treatment of comparatively deeper intrabony defects. These data apparently suggest an enhanced regenerative potential of the rhFGF-2/HA hydrogel reported in the present study. Further studies evaluating different carriers for rhFGF-2 and growth factor dosing may further clarify the relative regenerative potential of new formulations. A limitation of the present study was the fact that no positive control group treated with the HA carrier alone was employed. However, the scarce literature available strongly suggests that HA may possess little, if any, regenerative effect when employed alone in surgical therapy (Hunt et al 2001). Engström et al (2001) reported 0.5mm increase in bone height when HA was employed in conjunction with barrier membranes. Ballini et al (2009) reported PAL gains of 2.6mm measured 9 months after treatment with the use of HA in conjunction with autogenous bone grafting in a case series with nine patients. Considering that similar, or even better, results have been documented with the use of autogenous bone grafting alone in the treatment of periodontal defects (Hiatt & Schallhorn 1973; Froum et al 1976; Abolfazli et al 2008), potential positive effects of HA
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2 has been demonstrated in several pre-clinical animal studies (Oi et al 2009; Murakami et al 1999, 2003; Takayama et al 2001; Rossa et al 2003) with accelerated regeneration of cementum and functional periodontal ligament (Sato et al 2004; Nakahara et al 2004, Yang et al 2004). Local application of FGF-2 significantly enhances vascularization and osteogenesis, defect filling and cementum formation (Rossa et al 2003, Sato et al 2004, Nakahara et al 2004).
Therefore, FGF-2 is a potential candidate as a therapeutic molecule due to its anabolic effects on cementum, periodontal ligament and bone and may offer an exciting new alternative in periodontal regenerative surgery. The objective of this study was to evaluate if rhFGF-2 delivered into periodontal defects in a hyaluronic acid (HA) carrier would enhance the clinical parameters of regeneration of the periodontal attachment apparatus. The hypothesis under investigation was that the local application of rhFGF2/HA would significantly enhance periodontal attachment and bone gain in intrabony defects in humans.
Materials and Methods Study Population and Experimental Design The study was designed as a randomized, prospective, split-mouth, controlled clinical trial. It was conducted in accordance with the guidelines of the World Medical Association Declaration of Helsinki (version VI, 2002), after approval of the study design and consent by the Faculty of Medicine, Ethical Committee of Medical Research, Federal Fluminense University (Approval protocol CEP-HUAM/CCM 21/03). Written informed
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consent was obtained from all subjects. Systemically healthy patients with moderate to advanced chronic periodontitis referred to the Department of Periodontology, Faculty of Dentistry, Federal Fluminense University for periodontal treatment of advanced periodontitis were recruited for and treated in the study. Inclusion criteria of the study were: (a) adult subjects with chronic periodontitis presenting (b) radiographic evidence alveolar bone loss at the proximal aspect of the tooth, (c) intrabony defect deeper than 4mm, (d) probing pocket depth >6 mm at the site, (e) unremarkable general health according to medical history and clinical judgment, (f) no medications taken for at least six months and no antibiotics taken for twelve months before the beginning of the study. Subjects who have quit the habit of smoking for at least one year before the beginning of the study were considered as non-smokers and, if the other included criteria were met, were included in the present sample. Exclusion criteria for the study were: (a) subjects with “early onset” or aggressive forms of periodontitis, (b) current smokers, (c) presence of significant systemic diseases (i.e., cancer, AIDS, diabetes), (d) clinical evidence of furcation defects, (e) presence of apical radioleucency and (f) previous lack of cooperation with the maintenance program. Two defects were selected from each patient, and were randomly assigned, immediately before each surgical appointment, by computer generated random sequence allocation (custom randomisation software, Department of Biostatistics, Federal Fluminense University) to one of the two treatment modalities employed: (a) Control: open instrumentation of the root surfaces and bone defects via flap modified papilla preservation flap; (b) Test: open instrumentation of the root surfaces and bone defects via flap modified papilla preservation flap and application of a rhFGF-2/HA in the intrabony defect.
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Treatment allocation was registered in an allocation table sheet that was unavailable to the clinical examiner throughout the study. Both the control and test sites were treated at the same surgical appointment and no information on treatment allocation was provided to the patient. All surgical procedures were performed by a single operator (RBS), which became aware of the site allocation only after completed root planning and conditioning, and immediately before the application of the growth factor in the test sites. A strict surgical protocol emphasizing careful hard- and soft-tissue management, root debridement, wound stability and infection control was employed (Santana et al 2009; Miranda et al 2013). Nonsurgical infection control was performed in the context of cause- related periodontal therapy and consisted of oral hygiene instructions, scaling and root planing, tooth polishing and plaque control measures performed six months prior to regenerative surgery. Patients were re-evaluated three and six months after the completion of the nonsurgical cause-related periodontal therapy and enrolled following the later evaluation appointment.
Clinical Recordings Clinical parameters were assessed using the cemento-enamel junction (CEJ) or, when applicable, another defined landmark, as a fixed reference point. All measurements were recorded using an UNC #15 periodontal probe (PCP-UNC 15, Hu-Friedy, Chicago, IL, USA) by a blinded, trained and calibrated examiner (CMMS), unaware of the treatment provided at baseline and one year after treatment. Measurements were recorded to the nearest mm. Full mouth plaque score (FMPS) was recorded dichotomously as the percentage of total surfaces (four sites per tooth) (O’Leary et al. 1972). BOP was also
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Figure 2 – Representative bone defect treated by control therapy.
A- Baseline clinical view; B - Surgical view after flap elevation; C - Closure (buccal view); D- Clinical view one year after treatment; E- Baseline radiographic aspect; F -Radiographic result one year after treatment
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Flap Design Intracrevicular incisions were made and buccal and lingual mucoperiosteal flaps, including at least one tooth mesial and another distal the tooth being treated, were elevated by blunt dissection. Extreme care was taken to preserve the marginal gingiva and inter-dental tissue to achieve better closure of the treated sites. Thus, the defect-associated inter-dental papilla was accessed with papilla preservation flap techniques (Takey 1985, Cortellini et al. 1995, Cortellini et al. 1999) according to indications. A vertical releasing incision was performed at the mesial aspect of the flap whenever necessary to obtain optimized, tensionless, access to the surgical area.
Root and bone defect debridement The bone defect was meticulously debrided with surgical curettes and ultrasonic instruments with special effort to remove all the inflammatory granulation tissue, but with no intention to perforate the cortical bone walls. Thorough root planing was performed with hand (Hu-Friedy, IL, USA), rotary (Intensiv, Grancia, Switzerland) and ultrasonic instruments (Cavitron, Dentisply, USA), to remove subgingival plaque and calculus until all root surfaces attained a hard glassy surface. During the instrumentation, the flaps were slightly elevated, carefully protected with periosteal elevators, and frequently irrigated with saline.
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Root conditioning and rhFGF-2 application After instrumentation, the root surfaces were washed with saline solution to remove any remaining detached fragments from the defect and surgical field. Then, a freshly prepared tetracycline hydrochloride paste in saline was applied with gentle circular movements on the instrumented and dried root surfaces of both test and control groups for three minutes. After that, the defect area was carefully rinsed with saline. Finally, on the experimental sites, 0.2ml of a gel containing 4mg/ml rhFGF-2 in sodium hyaluronate MW 1.3 x 106 (Ossigel®, Orquest, CA, USA) was applied on the dried root surfaces and bone defect (Figure 1C). The flaps were then repositioned and tension-free primary flap closure was obtained using internal mattress, and single interrupted sutures (5.0 Ethicon Sutures, Johnson & Johnson, NJ, USA or Gore-tex Sutures, W.L Gore, AZ, USA). Representative clinical cases are presented in figures 1 and 2.
Post-Operative Treatment The patients were prescribed systemic antibiotic therapy consisting of 200mg of doxycycline***** the day before surgery, followed by 100 mg daily continued until the 20th day after surgery. No surgical dressing was used. The patients were instructed to continue their regular home hygiene care, except in the operated area, which was cleaned by means of gentle topical applications of 0.2% chlorhexidine gluconate (Perioxidin®, Lacer, Barcelona, Spain) in saturated cotton swabs. Patients were instructed not to brush or floss the surgical area for 4 weeks, when mechanical plaque control was re-instituted. The sutures were removed two weeks after surgery. Hyper-mobile teeth were splinted either before or immediately after the surgical procedure.
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aintenance Schedule Patients were enrolled in a stringent post-operative supportive care programme following surgery (Santana et al 2009). All patients were seen weekly during the first three months and bi-weekly for the next three months. Thereafter, the patients were seen once monthly for the last six-month period of the study. After 1 year, the patients were recalled and re-instructed on proper oral hygiene. Sites were rescaled when indicated.
Radiographic assessments Radiographs were taken with a parallel, standardized, technique at baseline (Eickholz et al. 2004). Radiographs were digitized and analysed using NIH Image Analysis software. The anatomical landmarks for the study included cementum-enamel junction (CEJ), alveolar crest (AC) and base of the defect (BD) (Schei et al. 1959). Radiographic defect depth was measured along the root surface, parallel to the root axis, as the distance between CEJ and BD.
Statistical Analyses Quantitative data were recorded as mean and standard deviations. No data points were missing. Sample size was determined by Power analysis, assuming a standard deviation of the difference of 0.5, which indicated that with a sample of 20 subjects, the study would have >90% power to detect a 1-mm difference in the primary outcome measure PAL gain between the two groups. In order to verify the normality of the data, the kurtosis and skewness curves were used. Because all data were considered to be normal for the parameters analysed, Student’s paired t-test was used for intra-group (baseline versus 12
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months) and inter-group
(test versus control) comparisons. The level of significance
set at 5% was used in all statistical comparisons performed in the software ProStat (Poly Software International, Pearl River, NY, USA) .
Results Thirty adult patients (11 male, 19 female), 39 to 66 year-old (mean age of 49.2 years), meeting the inclusion criteria were treated. Complete data from 60 intrabony defects distributed in 15 incisors (8 test and 7 controls), 13 canines (six test and 7 controls), 18 premolars (9 in each group) and 14 lower molars (seven in each group) were available for statistical comparisons. Post-hoc power calculations assuming µ1equal to 4.4, µ2 equal to 2.2, sigma (σ) equal to 0.4, an alpha value of 0.05 and a sample size (n) of 30, revealed that the study had 100% power to detect the differences in the primary outcome measure. Healing was uneventful for all treated cases in both patient groups. Soft tissues healed within normal limits and no significant visual differences were noted between the treatment groups. No event of malaise, fever or pain was reported or detected. No significant edema, erythema, ulceration or hematoma was noted. Patients maintained an adequate level of plaque control throughout the duration of the study, as evidenced by the FMPS (17.6±2.1) and BOP (19.3±2.3) measurements obtained one year after treatment. Data from baseline clinical (PD, REC, PAL, CEJ-BD), radiographic (JCE-BDX, JCECOX, INTRAX) and trans-surgical (BD, BC, INTRA) measurements from test and control teeth are presented in Table 1. Inter-group measurements did not demonstrate significant differences between the groups, demonstrating homogeneity of the patient popula-
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tion under study. Measurements of distance CEJ-BD obtained after elevation of a mucoperiosteal flap were not significantly different to those obtained by trans-gingival probing demonstrating the equivalence of either method do estimate bone crest relation to the CEJ. Data from clinical measurements (PD, REC, PAL, CEJ-BD) performed in teeth from Experimental and Control groups one year after surgery are presented in table 2. Intra-group evaluations demonstrated reductions in PD, PAL e CEJ-BD, as well as increased REC, demonstrating significant differences between results from baseline and one year after treatment. These results demonstrate that both therapies were effective in significantly reducing periodontal probing depths and bone defects and enhancing probing attachment levels. However, both groups resulted in significant increase in gingival recession at treated sites. Inter-group comparisons demonstrated that the magnitude of changes in PD, PAL and CEJ-BD were significantly bigger in the test group (table 2), suggesting a higher efficacy of the test treatment in enhancing the clinical parameters of wound healing evaluated in the present experimental patient cohort. A comparison of frequency distributions of post-treatment PAL gain and residual PD is presented in table 3. Data demonstrates that the majority (60%) of sites in the test group exhibited a significant amount (>4mm) of PAL gain while in the control group the majority (80%) of sites presented less than 2mm of attachment gain. Moreover, the test group also presented with all sites with less than 5mm of residual PD, with most sites (56%) with residual probing depths shallower than or equal to 4mm. In contrast, all sites in the control group presented with residual PD equal to or deeper than 5mm.
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Discussion This longitudinal, randomized, controlled clinical trial demonstrated that an innovative combination of bioactive substances composed of rhFGF-2/HA applied in periodontal intrabony defects results in significantly more PD reduction, PAL and bone gains, higher frequency of sites with shallower PD and 4mm or more PAL gains than the control group one year after treatment, suggesting that this new treatment modality based on the principles of tissue engineering may demonstrate enhanced clinical outcomes in comparison with the conventional treatment. To the best of our knowledge, this is the first report on the use of a composite rhFGF-2/HA hydrogel in periodontal regeneration. Several systematic reviews with meta-analysis have demonstrated that open flap debridement has been widely employed as the control group in the majority of the studies evaluating regenerative techniques in intrabony defects (Trombelli et al. 2002, Needleman et al. 2005, Esposito et al. 2009, Graziani et al 2012), demonstrating greater variability of the results obtained (Tu et al 2008), probably due to type of surgical technique, flap design, incision and suturing employed (Graziani et al 2012). In line with the results of previous studies in the literature, the magnitude of change in the clinical parameters evaluated were relatively modest in the control group and inferior to the regenerative technique evaluated in parallel (Trombelli et al. 2002, Needleman et al. 2005, Esposito et al. 2009, Graziani et al 2012). The experimental therapy resulted in significantly more PD reduction, PAL gain and bone gains than the traditional open flap instrumentation, clearly demonstrating the bioactivity of the material, and enhancement of the healing in the human intrabony defect environment. The magnitude of changes compares positively with previous results re-
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ported in the periodontal literature for the treatment of similar periodontal defects with other regenerative therapies, including membranes, bone grafts, enamel matrix proteins or growth factors such as rhPDGF or rhGDF (Trombelli et al. 2002, Mariotti 2003, Needleman et al. 2005, Esposito et al 2009, Graziani et al 2012, Susin & Wikesko 2013, Lee & Wikesjo 2014). Two previous studies have evaluated the effects of rhFGF-2 in a hydroxypropyl cellulose gel formulation (Kitamura et al 2008; Kitamura et al 2011) and reported a relatively modest increase in radiographic bone level (1.85 ± 1.75 mm) and PAL gain (2.18 ± 1.33 mm) 9 months after treatment of comparatively deeper intrabony defects. These data apparently suggest an enhanced regenerative potential of the rhFGF-2/HA hydrogel reported in the present study. Further studies evaluating different carriers for rhFGF-2 and growth factor dosing may further clarify the relative regenerative potential of new formulations. A limitation of the present study was the fact that no positive control group treated with the HA carrier alone was employed. However, the scarce literature available strongly suggests that HA may possess little, if any, regenerative effect when employed alone in surgical therapy (Hunt et al 2001). Engström et al (2001) reported 0.5mm increase in bone height when HA was employed in conjunction with barrier membranes. Ballini et al (2009) reported PAL gains of 2.6mm measured 9 months after treatment with the use of HA in conjunction with autogenous bone grafting in a case series with nine patients. Considering that similar, or even better, results have been documented with the use of autogenous bone grafting alone in the treatment of periodontal defects (Hiatt & Schallhorn 1973; Froum et al 1976; Abolfazli et al 2008), potential positive effects of HA
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alone could not be ascertained. It is also important to realize that the magnitude of the difference between the experimental groups of the present study, in favor of the test therapy, for PAL (2.6mm) and bone gains (2.3mm) were 4 to 5-fold higher than in the previous studies. Indeed, the PAL gains reported by Ballini et al (2009) for HA plus autogenous bone was very similar than the amount of PAL gain observed in the control group of the present study. Thus, taken together, these data suggest that the improved results observed in the present study may be attributed to the biological activity of FGF-2, and that HA, although acting as a potentially adequate carrier, apparently did not significantly influence the treatment outcomes. Future controlled clinical studies employing HA alone may be necessary to further clarify this issue. In the present report we used HA as the carrier for rhFGF-2 due to the wellestablished bi-directional biologic relationships between both substances. The binding affinity of FGF-2 to glycosaminoglycans (GAG) with resultant protection from exogenous protease digestion is well characterized (Coltrini et al 1993). On the other hand, FGF-2 positively regulates GAG biosynthesis, especially in the periodontal environment (Shimabukuro et al 2005, 2008). FGF-2 regulates the hyaluronan metabolism in PDL cells by significantly increasing HA production, specifically enhancing mRNA expression of the enzymes hyaluronan synthase 1 and 2 (Shimabukuro et al 2005) and enhancement of the production of membrane-bound heparan sulfate proteoglycan syndecan (Shimabukuro et al 2008). Therefore, FGF-2 and HA share important biological interactions and may present synergistic activities. Previous data suggest little possibility that FGF-2 will cause systemic adverse effects after topical application, since no FGF-2 entered the circulation, and that locally-
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applied FGF-2 was not associated with antibody production (Kitamura et al 2008; Kitamura et al 2011). In accordance, the present population did not demonstrate any significant local or systemic alterations following application of FGF-2, as documented locally by soft tissues healing within normal limits and no significant visual differences between the treatment groups (no significant edema, erythema, ulceration or hematoma). Systemically, no event of malaise, fever or pain was reported or detected. These preliminary findings demonstrated a positive clinical response of human periodontal intrabony defects treated with a regenerative hydrogel composed of rhFGF-2 and HA, in accordance with the principles of tissue engineering, and suggest that this approach may offer potential benefits for the treatment of such lesions. Further studies with other potential combined therapies are warranted to further evaluate the efficacy of this new regenerative material and may further improve the clinical benefits of rhFGF-2/HA in periodontal surgical therapy.
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Acknowledgements: The authors thank Dr James W. Poser, PhD and Orquest, Inc (Mountain View, CA, USA) for the generous gift of the rhFGF-2/HA hydrogel employed in the present study.
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Table 1 – Measurements obtained at baseline MEASUREMENT
CONTROL
TEST
DIFFERENCE
P
PD
9.5±1.5
9.7±1.9
0.2±0.1
0.25 NS
REC
0.9±0.7
0.8±0.6
0.1±0.1
0.28 NS
PAL
10.3±1.3
10.4±1.6
0.1±1.6
0.33 NS
CEJ-BD
11.6±1.3
11.8±1.2
0.2±0.1
0.30 NS
CEJ-BDX
11.2±1.1
11.5±1.3
0.3±0.1
0.13 NS
CEJ-COX
6.1±1.5
6.2±1.9
0.1±1.7
0.42 NS
INTRAX
5.1±1.3
5.3±1.2
0.2±1.2
0.53 NS
BD
12.2±1.5
12.5±1.0
0.3±0.7
0.67 NS
BC
6.9±1.2
6.8±1.6
0.2±1.3
0.65 NS
INTRA
5.3±1.3
5.7±1.2
0.4±1.3
0.28 NS
One Wall
0.7±1.2
0.9±1.1
0.2±1.1
0.77 NS
Two Wall
1.2±1.3
1.4±1.2
0.2±1.3
0.68 NS
Three Wall
3.3±1.0
3.4±1.1
0.3±1.0
0.57 NS
PD = Probing depth, REC = gingival recession, PAL = probing attachment level, CEJ-BD = probing distance between cement-enamel junction and base of bone defect; CEJ-COX = radiographic distance between the CEJ and bone crest; JCE-BDX = radiographic distance between CEJ and the base of the bone defect); INTRAX = radiographic distance between the base of the bone defect and the bone crest; BD = intra-surgical distance between cement-enamel junction and base of bone defect; BC = intra-surgical distance between the CEJ and bone crest; INTRA = intrasurgical distance between the base of the bone defect and the bone crest. Three, Two and One Wall = the depth of the 3-, 2-, and 1-wall sub-components of the bony defect in mm. Data presented as mean ± standard deviation. P = statistical probability. NS = non-significant.
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Table 2 – Inter- and intra-group measurements obtained one year after treatment. MEASUREMENT
CONTROL
TEST
DIFERENCE
P
PDi
9.5±1.5
9.7±1.9
0.2±0.1
0.25 NS
PD1
6.6±1.3
4.2±0.8
2.4±0.1
