Effects of enamel matrix derivative on vascular endothelial growth factor expression and microvessel density in gingival tissues of periodontal pocket: a comparative study

Volume 82 • Number 4

Effects of Enamel Matrix Derivative on Vascular Endothelial Growth Factor Expression and Microvessel Density in Gingival Tissues of Periodontal Pocket: A Comparative Study Simone Domenico Aspriello,*† Antonio Zizzi,‡ Liana Spazzafumo,§ Corrado Rubini,‡ Teresa Lorenzi,i Daniela Marzioni,i Pedro Bullon,¶ and Matteo Piemontese*†

Background: Vascular endothelial growth factor (VEGF) stimulates proliferation and migration of endothelial cells, and correlates with inflammatory resolution and periodontal tissue healing. Enamel matrix derivative (EMD) seems to stimulate soft tissue healing. Our aim was to assess if topical EMD application in an instrumented periodontal pocket could affect angiogenesis at the gingival level. Methods: A total of 56 periodontal sites in 28 patients were treated with a single session of comprehensive scaling and root planing under local anesthesia after recording the clinical attachment level (CAL). EMD gel in the test site or only the vehicle propylene glycol alginate in aqueous solution in the control site of the same mouth was applied onto the root surfaces and into the pocket and left in place for 3 minutes. After 48 hours, gingival biopsies were collected for histologic and immunohistochemical analysis for VEGF and CD34 (for microvessel density [MVD] count) antibodies. Statistical comparisons were performed by analysis of variance test. Results: Endothelial VEGF expression and MVD were statistically different in the test site compared to the control site. VEGF expression and MVD of the control site were not correlated with CAL, whereas the test site showed high correlations among CAL and endothelial VEGF or MVD. Conclusions: EMD induces proliferation and viability and angiogenesis of human microvascular cells. Recent clinical and histologic studies found EMD to be useful as an adjunct to scaling and root planing in single-rooted teeth. Our findings may help to understand the mechanisms involved in soft tissue healing, through the ability of EMD to increase angiogenesis at periodontal pockets. J Periodontol 2011;82:606-612. KEY WORDS CD34 antigen; chronic periodontitis; immunohistochemistry; periodontal pocket; tissues; vascular endothelial growth factor. * Department of Clinical Specialistic and Dental Sciences - Periodontology, Polytechnic University of Marche, Torrette, Ancona, Italy. † Italian National Research Center on Aging, Ancona, Italy. ‡ Department of Neurosciences, Section of Pathologic Anatomy and Histopathology, Polytechnic University of Marche. § MS Statistical Center, Italian National Research Center on Aging. i Department of Molecular Pathology and Innovative Therapies, Section of Anatomy, Polytechnic University of Marche. ¶ Periodontics Department, School of Dentistry, University of Seville, Seville, Spain.

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asculogenesis involves the de novo formation of blood vessels in which endothelial progenitor cells assemble to form vessels in early development. The primitive vessel network subsequently expands and remodels to form a more mature network via angiogenesis, a process in which new blood vessels sprout from existing blood vessels.1 Angiogenesis, an essential component of normal wound healing and repair in which endothelial cells and their precursors actively participate, 2 and facilitates the removal of debris and assists in the development of a granulation tissue framework for wound closure.3 The major angiogenic factors include fibroblast growth factors (initial angiogenic stimuli), plateletderived growth factor, and vascular endothelial growth factor (VEGF) (prolonged angiogenic stimuli). VEGF may contribute to the angiogenic stimuli in wounds by direct effects on proliferating and migrating

doi: 10.1902/jop.2010.100180

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Aspriello, Zizzi, Spazzafumo, et al.

J Periodontol • April 2011

endothelial cells or indirectly by effecting persistent vascular permeability at the level of existing microvessels, which occurs during the early phases of wound repair, theoretically allowing deposition of the fibrin-rich matrix necessary for cellular migration.4,5 Cetinkaya et al.6 found that VEGF expression significantly increased and positively correlated with the number of blood vessels in the healing stage of periodontal disease. Therefore, VEGF expression and neovascularization seem to be related to the resolution of inflammation and spontaneous healing of periodontal tissues.6,7 VEGF was reduced in untreated chronic inflammatory periodontal disease. 8 In tissues the degree of angiogenesis can be evaluated by microvessel density (MVD) using an antibody against CD34, a glycosylated transmembrane protein present on progenitor endothelial cells.9 Enamel matrix derivative (EMD) is a product purified from porcine enamel matrix protein extracts containing 90% amelogenins, with the remaining 10% composed primarily of proline-rich non-amelogenins, tuftelin, tuft protein, and serum proteins.10 It has the potential to stimulate periodontal tissue regeneration,11,12 improving the biologic activities of periodontal tissue cells. EMD has an enhancing effect on proliferation and viability and in vitro angiogenesis of human microvascular endothelial cells (HMVECs),13 directly by stimulating endothelial cells and indirectly by stimulating the production of VEGF by periodontal ligament (PL) cells.14 The purpose of this study is to assess if topical application of EMD in an instrumented periodontal pocket (test site) could affect the gingival expression of VEGF and CD34 after 48 hours compared to the effect of the vehicle propylene glycol alginate (PGA) in aqueous solution applied in another instrumented periodontal pocket (control site) of the same mouth and for the same amount of time. MATERIALS AND METHODS Study Patients A total of 28 patients (15 males and 13 females), aged 48 to 62 years, and histologically diagnosed with localized, severe, chronic periodontitis were analyzed in this study approved by the Ethical Committee of Polytechnic University of Marche, Torrette, Ancona, Italy. All patients were informed of the purpose of the study and provided written consent before inclusion in the study. The diagnosis of localized severe chronic periodontitis was made based on the presence of £30% of measured sites with clinical attachment level (CAL) ‡5 mm.15 The criteria for inclusion of patients and areas in this study were individuals who were non-smokers, free from systemic complications, and with no history of allergies. Subjects

had not used antibiotics in the 6 months before treatment, nor had they been treated for periodontitis during the previous 2 years, and they had radiographic and clinical evidence of two defects with a probing depth (PD) >5 mm, and osseous defect depth estimated from radiographic evaluation as >3 mm, in need of periodontal regeneration. Study Design The study was performed as an intraindividual, doublemasked, randomized placebo-controlled design comparing VEGF expression and MVD of two sites in the same mouth treated with EMD gel # (test) or with only a PGA carrier (control) before periodontal regeneration. All patients received oral hygiene instruction, fullmouth scaling and root planing performed under local anesthesia with articaine hydrochloride, 40 mg/mL, and (R)-adrenalin hydrochloride,** 0.012 mg/mL, with periodontal curets†† and occlusal adjustment when indicated. After scaling and root planing, the teeth were polished with three pastes with decreasing order of abrasiveness.‡‡§§ Reevaluation examinations were performed 2 months after initial therapy to determine patient response to therapy and to confirm the need for periodontal regeneration of the two different sites. Experimental study was initiated on patients when adequate plaque control, judged as 25%) and modest (positive cells 95%. Statistical Analyses Results are expressed by means – SD. Univariate analysis was performed by double-tail paired t test for continuous variables. A Pearson correlation analysis was used as function of CAL with endothelial, connectival, epithelial VEGF expression and MVD. The Pearson correlation coefficient (r) is a measure of the strength of the linear relationship between two variables. If the Pearson correlation coefficient r value is near – 1, there is a perfect correlation. Values of Pearson correlation r that lie between – 0.75 and – 1 showed a very high degree of correlation; between – 0.25 and – 0.75, high–moderate degree of correlation, between 0 and – 0.25, low degree of correlation; and Pearson correlation coefficient r value around zero means no correlation. Differences within groups in VEGF expression (endothelial, dermal, and epithelial) and MVD between test and control site were assessed by repeated measures analysis of variance (ANOVA) with one covariate (CAL). ANOVA for repeated measures is used when subjects are measured more than once to determine whether statistically significant change has occurred among multiple occasions. It was performed with a multivariate Wilk lamda statistic for testing the effect of topical application of EMD after 48 hours. Statistical analysis was performed with a statistical software program,¶¶¶ and a probability value