The International Journal of Periodontics & Restorative Dentistry © 2015 BY QUINTESSENCE PUBLISHING CO, INC. PRINTING OF THIS DOCUMENT IS RESTRICTED TO PERSONAL USE ONLY. NO PART MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM WITHOUT WRITTEN PERMISSION FROM THE PUBLISHER.

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Microvessel Density Evaluation of the Effect of Enamel Matrix Derivative on Soft Tissue After Implant Placement: A Preliminary Study George Furtado Guimarães, DDS, MSc, PhD1 Vera Cavalcanti de Araújo, DDS, MSc, PhD2 James Carlos Nery, DDS, MSc, PhD1 Daiane Cristina Peruzzo, DDS, MSc, PhD3 Andresa Borges Soares, DDS, MSc, PhD4 Enamel matrix derivative (EMD) is commonly used in periodontal therapy and has been used successfully for periodontal regeneration. In addition, this material has a possible angiogenic effect that has been associated with enhanced wound healing. The aim of this study was to evaluate the effect of EMD on microvessel density (angiogenesis) on the soft tissues surrounding newly placed implants after 14 days. Five patients were selected, each requiring at least one implant on each side of the maxilla, in a split-mouth experimental design. The implants were placed in a two-stage procedure. Each side was then randomized as test or control. On the test side, 0.1 mL of EMD was topically applied to the soft tissues surrounding the implants, while the control side did not receive any treatment. Second-stage surgery was performed after 14 days. A 6-mm punch biopsy was performed for each implant, with the samples subsequently prepared for histology and immunohistochemistry. Quantitative vascularization analysis was performed, which involved counting three areas or “hotspots” containing vessels strongly positive for CD34 and CD105, a pan-endothelial and new vessel marker, respectively. There was no significant difference between test and control groups when evaluating the formation of new blood vessels. The total number of blood vessels, however, was significantly higher in the group treated with EMD (test group). Within the limits of the present study, it can be concluded that topical application of EMD on the soft tissues surrounding newly placed implants resulted in an increased number of blood vessels at 14 days, suggesting that EMD may play a beneficial role in this aspect of wound healing. (Int J Periodontics Restorative Dent 2015;35:733–738. doi: 10.11607/prd.2044)

Lecturer and Researcher in Implantology, Department of Oral Pathology, São Leopoldo Mandic Institute and Research Center, Campinas, São Paulo, Brazil. 2Professor and Researcher in Pathology, Department of Oral Pathology, São Leopoldo Mandic Institute and Research Center, Campinas, São Paulo, Brazil. 3Lecturer and Researcher in Periodontology and Implantology, Department of Oral Pathology, São Leopoldo Mandic Institute and Research Center, Campinas, São Paulo, Brazil. 4Lecturer and Researcher in Pathology, Department of Oral Pathology, São Leopoldo Mandic Institute and Research Center, Campinas, São Paulo, Brazil. 1

Correspondence to: Dr Daiane Cristina Peruzzo, São Leopoldo Mandic Institute and Research Center Rua José Rocha Junqueira, 13 Ponte Preta 13045-755, Campinas, São Paulo, Brazil. Email: [email protected] ©2015 by Quintessence Publishing Co Inc.

The effectiveness of enamel matrix derivative (EMD) in improving clinical outcomes of periodontal treatment, mainly in angular and dehiscence bone defects, has been reported.1–5 Studies analyzing the effect of EMD in basic biological processes, such as cell proliferation, cell differentiation, chemotaxis, osteogenesis, cementogenesis, dentinogenesis, and angiogenesis have also been performed.6–11 Improved wound healing has been observed clinically, with EMD applied topically for instrumented pockets.12 Improved gingival tissue healing after EMD application has been related to its effect on endothelial cells.13 Angiogenesis is the process of new blood vessel formation from preexisting vasculature. This event plays a critical role in some physiological and pathological conditions, including wound healing, tissue repair, the menstrual cycle, cancer, and various ischemic and inflammatory diseases.14,15 In terms of the wound healing after tissue injury, new capillaries invading the fibrin clot are critical for the formation of early granulation tissue, as well as to deliver nutrients, oxygen, and inflammatory cells to the wound site.16 On the contrary, absence or deficiency of angiogenesis may result in impaired and/or delayed wound healing, which could lead to ulcer formation.17

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734 Given the importance of angiogenesis in wound healing, studies have analyzed the effects of EMD on angiogenesis. Animal models have been used to ascertain whether EMD could promote an angiogenic effect, consequently improving surgical wound healing, with significantly positive effects.11 Furthermore, the effect of EMD on human microvascular endothelial cells has been reported in an in vitro study, suggesting that EMD stimulates angiogenesis both directly and indirectly, via endothelial cell stimulation and by stimulating the production of angiogenic factors in periodontal ligament cells, respectively.13 The aim of this study, therefore, was to evaluate the effect of EMD on microvessel density (MVD; angiogenesis) in the soft tissues surrounding implant placement after 14 days.

Method and materials Sample selection

Five patients seeking dental implant surgery at the São Leopoldo Mandic Institute and Research Center (Campinas, Brazil) were selected. Inclusion criteria were (1) the requirement of at least one implant on each side of the maxilla, in a split-mouth manner; (2) good systemic health (ASAI); and (3) absence of periodontal diseases (gingivitis and periodontitis). The exclusion criteria were (1) the requirement of an additional procedure, such as sinus lifting/ grafting or guided tissue regeneration; (2) presence of diabetes; and

(3) smoking. Before treatment, the patients provided written informed consent. All procedures were approved by the research ethics committee of the São Leopoldo Mandic Institute and Research Center (no. 2011/0057).

Experimental procedures

Each side of the maxilla was randomly assigned as test or control, to receive topical application of EMD (Emdogain, Biora) on all tissues surrounding the implant or to not receive any treatment, respectively. All tissues were carefully manipulated.

Surgical and maintenance procedures

All procedures were performed by the same expert operator (G.F.G.). After local anesthesia, incisions and flap design were performed in the same manner for both test and control sides (Fig 1a). After a full-thickness flap, a Straumann bone level SLActive implant (Straumann) was placed in a two-stage procedure. Immediately after implant placement, the test side received 0.1 mL of EMD, topically applied on the cover screws, exposed bone structures, and all surrounding epithelial and connective tissues (Fig 1b). The control side did not receive any treatment. Before suturing, photos were taken and measurements recorded using the adjacent teeth to determine the exact implant position and facilitate implant location

at the second stage. After suturing, the test side received a second topical application of 0.1 mL EMD over the suture. Follow-up assessment was performed on days 5 and 10 via clinical examination and suture removal, respectively. The second surgical stage was performed 14 days later. At this time, each implant position was determined according to the location references given before suturing, as described earlier. A 6-mm punch biopsy (Miltex) was performed for each implant. Samples were marked with ink in situ to orient histologic sections (Fig 1c). The samples were then carefully removed (Fig 1d) and placed in 10% buffered formalin. After 21 days, the patients were referred for prosthetic restorations, as is commonly done for this type of implant.18

Histologic procedures and immunohistochemistry

Sections measuring 5 μm were obtained from formalin-fixed, paraffinembedded samples and routinely stained with hematoxylin and eosin. Sections measuring 4 μm were pretreated with organosilane for immunohistochemistry. The sections were deparaffinized and hydrated, and endogenous peroxidase activity was quenched by immersion in 3% hydrogen peroxidase. Antigen retrieval was achieved by boiling in a steamer immersed in citrate buffer (pH 6.0) or pepsin, according to the antigen used. Sections were incubated at 37ºC with a serum-free protein block (code X0909, Dako).

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735 Subsequently, the sections were incubated with the CD34 and CD105 antibodies, with amplification performed with the Advance system (Code K4067, Dako). The sections were then stained with diaminobenzidine tetrahydrochloride and counterstained with hematoxylin (Table 1).

a

b

c

d

MVD assessment

Immunohistochemistry for CD34 and CD105 was interpreted by two expert examiners (V.C.A. and A.B.S.), who were blinded to the treatments. The most vascularized areas or hotspots for each case were chosen at low magnification. To assess vessel number, images were obtained from three magnification fields per case (40× objective, 0.44-mm field diameter) using a charge-coupled device camera adapted to an Olympus CX30 microscope and analyzed with Imagelab analysis software (version 2.4), which allows manual segmentation of the target vessels. Microvascular blood density for CD34 and CD105 was defined as the mean number of microvessels counted. There was no restriction on the size of a microvessel; however, vessels with muscular walls were not included.

was used to compare the different groups (test and control), via analysis of the CD34 and CD105 hotspot vessel counts. All analyses were performed using SPSS20 software (SPSS), with a significance level of .05.

Statistical analysis

Results

Data were described in terms of the mean, median, minimum, and maximum values and the standard deviation (SD). The Wilcoxon test

Clinical results were evaluated, though they were not the objective of this study. After 14 days, good healing was observed at both sites,

Fig 1    (a) Incision and flap design for test and control sides. (b) Topical application of EMD on the cover screw; exposed bone structure and epithelial and connective tissues surrounding the implants. Punch biopsies and marked samples (c) and sample removal (d) at second-stage surgery.

Table 1 Details of the antibodies used for immunohistochemistry Specificity

Clone

Dilution

Source

Buffer (AR)

CD34

QBEnd 10

1:50

Dako

Citrate

CD105

SNG

1:10

Dako

Pepsin

AR = antigen retrieval.

more markedly on the test side. All patient-centered outcomes were reviewed, with no postoperative complications seen during the study period. In terms of MVD for CD105, a low number of small positive vessels were detected in both the test (mean = 4; median = 3; maximum = 9; minimum = 1; SD = 3) and control (mean = 3; median = 2; maximum = 5; minimum = 2; SD = 2) groups. However, no significant difference was observed (P = .6367) (Fig 2).

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10

Fig 2    Box and whisker plots of microvessel density in the control and test groups, using CD105 and CD34 antibodies, showing the median, minimum, and maximum values.

70 60

8 6

MDV

MDV

50

4

40 30 20

2 0

10 Control group

EMD

0

Control group

CD105

EMD

CD34

Fig 3    Immunostaining of blood vessels on the control and test sides. Few CD34positive blood vessels are seen on the control side (left), while an increase in positive vessels is observed on the test side (right).

In terms of MVD for CD34, high vascularity was observed in both the test (mean = 48; median = 43; maximum = 64; minimum = 38; SD = 11) and control (mean = 36; median = 36; maximum = 46; minimum = 23; SD = 9) groups, with values being significantly higher in the test group (P = .0313; Figs 2 and 3).

Discussion The use of EMD has demonstrated some advantages over other methods for regenerating periodontal

tissues, mainly in intrabony defects. This product has been used widely in the clinical setting for this purpose, but its properties and biological mechanisms are poorly understood.19 Clinical trials have shown that EMD topically applied for regenerative therapy of bone defects could improve the early healing of periodontal soft tissue wounds.12,20 EMD is thought to induce proliferation, migration, adhesion, mineralization, and differentiation of cells in periodontal tissues,21 and also appears to control immune cell–induced inflammation.22

Wound healing is a complex process that involves cell migration, attachment, and proliferation, with many of these processes being controlled by cytokines and growth factors.23 Angiogenesis has been accepted as influencing the wound healing process. Following an injury, the capillaries that invade the fibrin clot are extremely important in the formation of early granulation tissue, because of the delivery of nutrients, inflammatory cells, and oxygen to the wound site.14,16 It has been shown that EMD increases endothelial cell proliferation and

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737 chemotaxis, as well as the rate of new blood vessel formation, therefore, supporting the hypothesis that the use of EMD in the clinical setting could stimulate angiogenesis and accelerate the healing process.13 Angiogenesis can be assessed via MVD.24 In this study, the endothelial markers chosen for assessing MVD were CD34 and CD105. CD34 is considered a panendothelial marker,25 and CD105 stains proliferating endothelial cells26 strongly in newly formed blood vessels, whereas preexisting vessels are negative.27 Schlueter et al13 used an in vitro assay for angiogenesis to assess the quantity of newly formed vessels in cultured human microvascular endothelial cells 6 hours after EMD application. New vessel formation occurred faster in the presence of EMD (test group) compared with the control group (P < .05). In the present study, the number of newly formed vessels, stained using CD105, was not significantly different in the test and control groups (P = .6367), with both groups showing a low number. One possible explanation for the CD105 results is that most newly formed blood vessels have reached maturation after 14 days, which was the time between implant placement and biopsy collection. This fact, however, does not exclude the possibility that EMD application may increase new vessel formation at the initiation of angiogenesis. In an in vivo study using a murine model and immunohistochemistry (CD31), Yuan et al11 confirmed significantly more blood vessels surrounding collagen implants on

EMD-treated specimens compared with the control group. This was corroborated by the present study, in which significantly higher MVD was observed in the EMD group. CD34 assessment for the total number of blood vessels after 14 days showed a significantly higher value for the test group than for the control group. This study hypothesized that the increased number of blood vessels observed in the test group was because of an increase in vascular endothelial growth factor (VEGF) production by gingival fibroblasts secondary to EMD application on the soft tissues surrounding implants; this was because the principal mechanism that supports positive effects of EMD on endothelial cells is based on its ability to regulate fibroblast-produced VEGF.13 Transforming growth factor beta (TGF-β) is another important growth factor that promotes angiogenesis. TGF-β1 has been shown to promote both in vivo28 and in vitro angiogenesis.29 A threefold increase in the production of TGF-β1 was observed in periodontal ligament cells stimulated by EMD,30 and a 2.5- to 3.8-fold increase in TGF-β1 production by periodontal ligament cells has also been reported.13 Therefore, the positive angiogenic effect of EMD observed in this study may, in part, be the result of these growth factors. Dental implant surgery may require excessive manipulation of the soft tissues; therefore, healing around implants may be compromised. In addition, the influence of systemic disease can lead to delayed or impaired healing. The

possible angiogenic effect of EMD could therefore benefit wound healing in certain cases. To the authors’ knowledge, this is the first study aimed at evaluating the effect of EMD on MVD (angiogenesis) in surrounding soft tissues after implant placement. The results of the present study suggest that topical application of EMD on the soft tissues surrounding newly placed implants increases vascularization at 14 days, proposing that EMD may play a beneficial role in wound healing. Although only a single indicator, blood vessel formation is paramount to the process of tissue repair. Nevertheless, additional studies are necessary to explore these findings and to evaluate the role of other important factors involved in the wound healing process. Further clinical trials are therefore recommended to elucidate the relationship between EMD and optimized wound healing.

Conclusions Within the limits of the present study, it can be concluded that topical application of EMD on the soft tissues surrounding newly placed implants resulted in an increased number of blood vessels at 14 days, suggesting that EMD may play a beneficial role in this aspect of wound healing.

Acknowledgments The authors reported no conflicts of interest related to this study.

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738 References  1. Hammarström L, Heijil L, Gestrelius S. Periodontal regeneration in a buccal dehiscense model in monkeys after application of enamel matrix proteins. J Clin Periodontol 1997;24(9 Pt 2):669–677.  2. Heijil L, Heden G, Svardstrom G, Ostgren A. Enamel matrix derivative (Emdogain®) in the treatment of intrabony periodontal defects. J Clin Periodontol 1997;24(9 Pt 2):705–714.  3. Francetti L, Trombelli L, Lombardo G, et al. Evaluation of efficacy of enamel matrix derivative in the treatment of intrabony defects: A 24-mouth multicenter study. Int J Periodontics Restorative Dent 2005;25(5):461–473.   4. Heden G, Wenntrom J. Five-year followup of regenerative periodontal therapy with enamel matrix derivative at sites with angular bone defects. J Periodontol 2006;77:295–301.   5. Sculean A, Chiantella GC, Arweiler NB, Becker J, Schwarz F, Stavropoulos A. Five-year clinical and histologic results following treatment of human intrabony defects with an enamel matrix derivative combined with a natural bone mineral. Int J Periodontics Restorative Dent 2008;28:153–161.  6. Gestrelius S, Anderson C, Johansson A, Persson E, Brodin A, Rydhag L, et al. Formulation of enamel matrix derivative for surface coating. Kinetics and cell colonization. J Clin Periodontol 1997;24 (9 Pt 2):678–684.  7. Hoang A, Oates T, Cochran D. In vitro wound healing responses to enamel matrix derivative. J Periodontol 2000;71: 1270–1277.   8. Cattaneo V, Rota C, Silvestri M, Piacentini C, Forlino A, Gallanti A, et al. Effect of enamel matrix derivative on human periodontal fibroblasts: Proliferation, morphology and root surface colonization. An in vitro study. J Periodontal Res 2003; 38:568–574.   9. Zeldich E, Koren R, Dard M, Nemcovsky C, Weinreb M. Enamel matrix derivative protects human gingival fibroblasts from TNF-induced apoptosis by inhibiting caspase activation. J Cell Physiol 2007;213(3):750-758.

10. Kaida H, Hamachi T, Anan H, Maeda K. Wound healing process of injured pulp tissues with emdogain gel. J Endod 2008; 34(1):26–30. 11. Yuan K, Chen C, Lin M. Enamel matrix derivative exhibits angiogenic effect in vitro and in a murine model. J Clin Periodontol 2003;30(8):732–738. 12. Wennstrom J, Lindhe J. Some effects of enamel matrix proteins on wound healing in the dento-gingival region. J Clin Periodontol 2002;29(1):9–14. 13. Schlueter S, Carnes D, Cochran D. In vitro effects of enamel matrix derivative on microvascular cells. J Periodontol 2007; 78(1):141–151. 14. Li J, Zhang Y, Kirsner R. Angiogenesis in wound repair: Angiogenic growth factors and the extracellular matrix. Microsc Res Tech 2003;60(1):107–114. 15. Dvorak h. Angiogenesis: Update 2005. J Thromb Haemost 2005;3(8):1835–1842. 16. Tonnesen M, Feng X, Clark R. Angiogenesis in wound healing. J Investig Dermatol Symp Proc 2000;5(1):40–46. 17. Folkman J, Shing Y. Angiogenesis. J Biol Chem 1992;267(16):10931–10934 18. Morton D, Bornstein M, Wittneben J, et al. Early loading after 21 days of healing of nonsubmerged titanium implants with a chemically modified sandblasted and acid-etched surface: Two-year results of a prospective two-center study. Clin Implant Dent Relat Res 2010;12(1):9–17. 19. Cattaneo V, Rota C, Silvestri M, et al. Effect of enamel matrix derivative on human periodontal fibroblasts: Proliferation, morphology and root surface colonization. An in vitro study. J Periodontal Res 2003;38(6):568–574. 20. Tonetti M, Fourmousis I, Suvan J, Cortellini P, Brägger U, Lang N. Healing, post-operative morbidity and patient perception of outcomes following regenerative therapy of deep intrabony defects. J Clin Periodontol 2004;31(12): 1092–1098. 21. Izumi Y, Aoki A, Yamada Y, Kobayashi H, Iwata T, Akizuki T, et al. Current and future periodontal tissue engineering. Periodontol 2000 2011;56(1):166–187.

22. Fujishiro N, Anan H, Hamachi T, Maeda K. The role of macrophages in the periodontal regeneration using Emdogain gel. J Periodontal Res 2008;43(2): 143–155. 23. Rincon J, Haase H, Bartold P. Effect of Emdogain on human periodontal fibroblasts in an in vitro wound-healing model. J Periodontal Res 2003;38(3): 290–295. 24. Sharma S, Sharma M, Sarkar C. Morphology of angiogenesis in human cancer: A conceptual overview, histoprognostic perspective and significance of neoangiogenesis. Histopathology 2005; 46(5):481–489. 25. Barresi V, Cerasoli S, Vitarelli E, Tuccari G. Density of microvessels positive for CD105 (endoglin) is related to prognosis in meningiomas. Acta Neuropathol 2007;114(2):147–156 26. Liorca O, Trujillo A, Blanco F, Bernabeu C. Structural model of human endoglin, a transmembrane receptor responsible for hereditary hemorrhagic telangiectasia. J Mol Biol 2007;365(3):694–705 27. Kumar P, Wang J, Bernabeu C. CD 105 and angiogenesis. J Pathol 1996;178(4): 363–366. 28. Roberts A, Sporn M, Assoian R, et al. Transforming growth factor type beta: Rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. Proc Nati Acad Sci USA 1986; 83(12):4167-4171. 29. Iruela-Arispe M, Sage E. Endotheial cells exhibiting angiogenesis in vitro proliferate in response to TGF-beta1. J Cell Biochem 1993;52(4):414–430. 30. Van der Pauw M, Van den Bos, Everts V, Beertsen W. Enamel matrix-derived protein stimulates attachment of periodontal ligament fibroblasts and enhances alkaline phosphatase activity and transforming growth factor beta1 release of periodontal ligament and gingival fibroblasts. J Periodontol 2000;71(1):31–43.

The International Journal of Periodontics & Restorative Dentistry © 2015 BY QUINTESSENCE PUBLISHING CO, INC. PRINTING OF THIS DOCUMENT IS RESTRICTED TO PERSONAL USE ONLY. NO PART MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM WITHOUT WRITTEN PERMISSION FROM THE PUBLISHER.

Microvessel Density Evaluation of the Effect of Enamel Matrix Derivative on Soft Tissue After Implant Placement: A Preliminary Study.

Enamel matrix derivative (EMD) is commonly used in periodontal therapy and has been used successfully for periodontal regeneration. In addition, this ...
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