Effects of epigallocatechin-3-gallate on the healing of extraction sockets with a periapical lesion: A pilot study in dogs Ji-Youn Hong,1* Jeyoung Yon,2* Jung-Seok Lee,2 In-Kyeong Lee,2 Cheryl Yang,2 Min-Soo Kim,2 Seong-Ho Choi,2 Ui-Won Jung2 1

Department of Periodontology, School of Dentistry, Kyung Hee University, Seoul 134-727, Republic of Korea Department of Periodontology, Research Institute for Periodontal Regeneration, College of Dentistry, Yonsei University, Seoul 120-752, Republic of Korea

2

Received 6 February 2014; revised 29 May 2014; accepted 5 June 2014 Published online 17 July 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33238 Abstract: The aim of this study was to characterize the healing process of extraction sockets with a periapical lesion following immediate graft with collagenated bovine bone mineral (CBBM) soaked with epigallocatechin-3-gallate (EGCG). Following induction of periapical lesions in premolars in five dogs, treatment of extraction sockets was divided into three groups: control (no treatment) and two test groups, CBBM with or without EGCG. 3D reconstruction and superimposition of the digital images were used to measure the dimensional changes in alveolar ridge. Histologic specimen was evaluated in all groups. The horizontal ridge widths at 4mm level were wider in both test groups (3.3 6 1.7 mm in CBBM; 3.0 6 1.7 mm in CBBM1EGCG) than in the control

group (1.7 6 2.4 mm). Fibrosis and limited new bone formation were observed in the apical regions of test groups; however, the extent of fibrosis was less in the CBBM1EGCG group. Within the limitations of this study, it was conjectured that adjunctive use of EGCG with CBBM can be a candidate biomaterial in grafting of extraction socket with periapical lesion. Bone regeneration at the coronal region of the CBBM grafted socket might not be influenced by the presence of a C 2014 Wiley Periodicals, Inc. J Biomed Mater Res periapical lesion. V Part B: Appl Biomater, 103B: 727–734, 2015.

Key Words: bio-oss collagen, epigallocatechin gallate, histology, periapical periodontitis, tooth extraction

How to cite this article: Hong J-Y, Yon J, Lee J-S, Lee I-K, Yang C, Kim M-S, Choi S-H, Jung U-W. 2015. Effects of epigallocatechin-3-gallate on the healing of extraction sockets with a periapical lesion: A pilot study in dogs. J Biomed Mater Res Part B 2015:103B:727–734.

INTRODUCTION

Extraction sockets with intact thick bony walls naturally fill up with bone via a series of healing processes in the absence of any grafting. However, such an ideal situation is rarely encountered in the clinic, as many indications for tooth extraction are associated with severe breakdown of the surrounding alveolar bone. Uncontrolled endodontic infection is one of the well-known reasons for tooth extraction. The pathogenesis of the disease is pulpal necrosis from bacterial infection, which may induce an inflammatory response and bone resorption at the periapical area of the tooth.1,2 Although the radiographic features of periapical translucencies appear similar, the histological features of the lesions vary from periapical granulomas to abscesses and cysts.3,4 The presence of inflammatory cells, proinflammatory cytokines, necrotic materials, and bacteria in the lesion could be risk factors for compromised outcomes after the subsequent placement of foreign materials such as dental implants or bone substitutes.5 Extraction sockets with previous apical lesions are conventionally left to heal naturally

for several months to allow the resolution of any remaining sources of infection. Delayed surgical procedures are associated with the problem of dimensional changes in the hard and soft tissues during the healing period of extraction socket, as described in many clinical reports.6,7 Significant reductions of the alveolar ridge in both the horizontal and vertical directions have been observed over a 6-month healing period, with the change being more pronounced on the buccal aspect.8,9 The atrophied ridge tends to have an inadequate volume of buccolingual bone for the placement of dental implants, resulting in an esthetically undesirable prosthesis. This has led to the proposal of ridge preservation to maintain a stable preexisting tissue envelope volume.10 To this end, the grafting of various bone substitutes into fresh extraction sockets has been reported, including collagenated bovine bone mineral (CBBM; Bio-Oss Collagen, Geistlich Pharma, Wolhusen, Switzerland), which is comprised of deproteinized bovine bone mineral (Bio-Oss, Geistlich Pharma) and 10% highly purified porcine collagen type I.11–13 This CBBM appeared to

*Both authors contributed equally to this study. Correspondence to: U.-W. Jung (e-mail: [email protected]) Contract grant sponsor: Korea Health technology R&D Project, Ministry of Health & Welfare, Republic of Korea; contract grant number: A120822

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preserve the ridge dimensions by altering the tissue modeling process and delaying the healing process while being incorporated into the tissue. Surgical protocols recommend thorough debridement of the socket and systemic antibiotics to ensure that the tissue is free of any remaining infection.4 There have been controversial reports of the immediate implantation or guided bone regeneration in defects with periapical pathologies that some authors suggested favorable results,4,14,15 while others demonstrated undesirable outcomes including failed osseointegration and marginal bone loss.16–18 Furthermore, case reports of retrograde peri-implantitis have suggested that chronic periapical lesions from endodontic infection provide bacteria that colonize and contaminate implants that are placed into or near the extraction socket site even after long periods of healing, which showed the possibilities of long lasting sources of infection.19,20 Therefore, it is clear that methods for preventing reinfection and controlling inflammation are required to guarantee a stable outcome. Epigallocatechin-3-gallate (EGCG) is a major green-tea catechin that comprises the greater part of the polyphenolic compounds released when tea leaves are steamed. EGCG is reported to have antioxidative, anti-inflammatory, antibacterial, and anticarcinogenic effects due to the biologic benefits of catechin.21,22 In vitro studies have shown that EGCG may inhibit the lipopolysaccharide-induced production of inflammatory cytokines and the formation of osteoclasts, induce apoptosis, and increase the mineralization of bone and osteoblast differentiation.23–25 Although the pharmaceutical use of EGCG in the control of inflammation and suppression of bacterial disease in bone is receiving increased attention, the research findings are as yet mostly limited to laboratory studies. The aim of this study was to demonstrate the healing process of extraction sockets treated with grafting materials in circumstances in which infection sources possibly remained and to evaluate the biologic activity of EGCG when applied in addition to grafting materials, in a condition of induced periapical lesion in sites treated without any debridement. We hypothesized the adjunctive use of EGCG would decrease inflammatory response and severe breakdown of alveolar bone even in the infectious socket immediately grafted with CBBM, while the use of CBBM alone would be associated with pronounced destruction. MATERIALS AND METHODS

Animals The objects of this study were the extraction sockets of the mandibular second premolars from both right and left quadrants of five male beagle dogs (aged 15 months, typically weighing 10–15 kg). A mandibular second premolar of a beagle dog is two-rooted tooth, which has good divergence between mesial and distal roots to form two separate sockets. One two-rooted premolar per mandibular quadrant was extracted. Consequently, total of 20 sockets were created after extraction in five dogs. The animals initially had intact dentition and a healthy periodontium. The animal selection and management, surgical protocol, and preparation followed routines approved by the Institutional Animal

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Care and Use Committee, Yonsei Medical Center, Seoul, Korea (2011-0038). Surgical procedures All surgical procedures were performed in a sterilized operating room with the dogs under general anesthesia induced by intravenous administration of atropine (0.04 mg/kg; Kwang Myung Pharm., Seoul, Korea) and intramuscular injection of xylazine (Rompun, Bayer Korea, Seoul, Korea) combined with ketamine (Kerata, Yuhan, Seoul, Korea), followed by inhalation anesthesia (Gerolan, Choongwae Pharma, Seoul, Korea). Local infiltration anesthesia with 2% lidocaine HCl with epinephrine 1:100,000 (Kwang Myung Pharm, Seoul, Korea) was used at the intraoral surgical sites. Prior to the procedures, scaling was performed in all dentition and dental plaque was collected using a periodontal curette. After removing crown portion of the tooth with high-speed fissure bur and extirpation of dental pulp, the dental plaque was applied to the exposed root canal to facilitate pulpal infection.26 Periapical lesion was induced for 16 weeks and standard periapical radiograph was taken to confirm its presence. After 16 weeks, a silicone impression (Aquasil Monophase and Aquasil XLV, Dentsply Caulk, Milford, DE) was taken before surgery (defined as S0), then root extraction and bone grafting into the sockets were performed. A crevicular and two vertical incisions on the buccal aspect were made around the remaining mandibular second premolar and a mucoperiosteal flap was reflected. The tooth was sectioned along its midline using a high-speed carbide bur at the furcation, and a dental elevator was placed between the sectioned segments and rotated gently to complete separate the mesial and distal roots. The extraction was completed by careful application of an elevator and forceps to avoid surgical trauma on the crestal and septal bone around the roots. There was no surgical curettage and debridement of the infected periapical tissue after extraction. Four extraction sockets per animal were made and clinical measurements of the horizontal thickness of the buccal bone plate, lingual bone plate, and buccolingual width of the ridge were done with a caliper at 1 mm below the crest of the extraction socket. The sites were divided into three treatment groups: one control (with no treatment to the socket) and two test sites that received CBBM grafts in the socket either with EGCG (CBBM1EGCG) or without EGCG (CBBM). The CBBM was shaped using surgical scissors into a 3 3 3.5 3 2.5-mm3 cuboid to fit within the height of the socket and not exceed the alveolar crest margin. In this study, 0.5 mM EGCG (Sigma, St. Louis, MO) was diluted in distilled water, each CBBM piece was soaked in 0.8 mL of the solution. The effects of EGCG on infected socket lesions were evaluated using the split-mouth design. Distal extraction sockets were used for the two types of experimental treatments: CBBM in the left mandibular premolar and CBBM with EGCG in the right mandibular premolar. Both of the mesial extraction sockets were used as control sites for comparing the healing processes with the sites treated by socket grafting. In the experimental groups, the CBBM was placed into the socket without overfilling, visually ensuring that the material was saturated with blood. The periosteal flap was

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FIGURE 1. Schematic diagrams of the parameters used for the measurement of dimensional changes. S0, soft-tissue outline at preextraction; S4, soft-tissue outline at 4 weeks post-extraction; H4, alveolar ridge outline at 4 weeks post-extraction; green dotted line, long axis of the extraction socket; G0, reference line connecting the buccal and lingual gingival margins; VS, vertical distance between S0 and S4; VH, vertical distance between S0 and H4. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.].

positioned coronally to obtain primary closure, and interrupted sutures were placed using a resorbable material (Vicryl 5.0, Ethicon, Somerville, MA). Postsurgical management involved antibiotics (Cefazoline sodium 20 mg/kg, i.m.; Yuhan, Korea) daily for 3 days, a soft diet, and topical application of 0.2% chlorhexidine solution (Hexamedin, Bukwang Pharmaceutical, Seoul, Korea) for infection control. The sutures were removed at approximately 10 days post-extraction. After 4 weeks of healing, the dogs were euthanized by overdose with an intravenous injection of pentobarbital sodium (90– 120 mg/kg; Yuhan, Korea) and another silicone impression was taken (defined as S4).

was used to analyze the hard-tissue contour at 4 weeks (defined as H4). 3D-reconstructed views of the S0, S4, and 3D H4 profiles were superimposed using a software tool (Rapidform 2006, Inus Technology, Seoul, Korea) with the canines and third and fourth premolars as references. Several reference points were established to measure the changes in vertical and horizontal dimensions (Figure 1). In the outline of S0, two reference points that marked the buccal and lingual crevices of the decoronated crown were detected and a reference projection line (G0) was drawn between these points. The long axis of the extraction site was also marked. The following parameters were used to assess the vertical changes: VS: vertical discrepancy in the buccal or lingual aspect between S0 and S4 on reference line G0, which shows the change in soft-tissue dimensions between two time points. VH: vertical distance in the buccal or lingual aspect between S0 and H4 at reference line G0. In measuring the horizontal changes, several reference lines parallel to G0 and positioned apically at 4, 5, 6, 7, and 8 mm were marked. The buccal and lingual distance from the long axis of the extraction site was measured on each parallel line. The buccal plate located buccally from the long axis yielded positive values, which is also the same with lingual plate in the lingual direction. The width of the alveolar ridge on each reference line was also measured.

Histologic preparation Block sections of the surgical sites in both quadrants were immersed in 10% neutral buffered formalin for 10 days, decalcified with 5% formic acid, and then dehydrated in graded ethanol series. After paraffin embedding, serial 5mm-thick sections were cut at 80-mm intervals in the mesiodistal plane from the center of the extraction sockets, and stained with Masson’s trichrome.

Histologic and histomorphometric analysis General histologic observations were made with the aid of light microscopy (Olympus Research System Microscope BX51, Olympus, Tokyo, Japan) and a PC-based image-analysis system (Image-Pro Plus, Media Cybernetic, Silver Spring, MD; Adobe Photoshop CS4, Adobe Systems, San Jose, CA). In each histologic specimen, the coronal border of the socket was determined by projecting a line between the crests of the mesial and distal walls. The socket was divided into the following three parts with the same vertical lengths: coronal, middle, and apical. In photomicrographs taken at a magnification of 403, proportions of newly formed bone (NB), residual biomaterial (RB), and the connective tissue space (CTS) within the socket were measured [Figure 5(a)]. The same parameters within the region of interest (ROI) selected from each separate part (coronal, middle, and apical) were evaluated from the photomicrographs taken at a magnification of 1003 [Figure 5(b)]. The number of multinucleated cells in each part of the socket was also counted as a secondary outcome measurement in photomicrographs at 1003 magnification. All the data are expressed as mean 6 SD values.

Volumetric analysis A dental cast was fabricated from the impressions taken pre-extraction (i.e., S0) and at 4 weeks post-extraction (i.e., S4). The casts were scanned with the aid of a 3D laser scanner (VIVID 9i, Konica Minolta, Osaka, Japan) and a 3D softtissue profile was reconstructed. Micro-computed tomography (micro-CT) (SkyScan 1076, SkyScan, Kontich, Belgium)

Statistics Similar size and shape of the lesion were shown in the periapical view of every infected tooth, although the equal formation of apical abscess and relevant bone resorption was not ensured. The primary variables for the vertical dimensional changes were VS and VH at the buccal and lingual aspects. For horizontal dimensions, the ridge widths at 4, 5,

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TABLE I. Vertical Discrepancies of the Soft Tissue Dimensions Between Pre-extraction and 4 Weeks Post-extraction (Mean6 Standard Deviation; mm; n 5 5) VS (S0–S4) B Control CBBM CBBM1EGCG

VH (S0–H4) L

a

2.3 6 0.7 2.8 6 0.7a 2.5 6 1.0a

1.6 6 0.6 2.0 6 0.6 1.5 6 0.3

B

S4–H4 L

a

5.4 6 1.1 5.5 6 1.1a 4.6 6 0.6a

3.6 6 0.7 3.9 6 0.4 3.4 6 0.5

B

L a

3.1 6 0.7 2.7 6 0.6a 2.1 6 0.8a

1.9 6 0.5 1.9 6 0.7 1.9 6 0.3

S0, soft tissue outline pre-extraction; S4, soft tissue outline 4 weeks post-extraction; H4, alveolar ridge outline 4 weeks post-extraction; VS, vertical distance between S0 and S4; VH, vertical distance between S0 and H4; B, buccal; L, lingual. a Statistical significant difference between buccal and lingual aspect in each group (p < 0.05).

6, 7, and 8 mm apical from reference line G0 were evaluated. The number of multinucleated cells was counted as a secondary outcome. The Kolmogorov–Smirnov test revealed that the distribution was not detectably nonnormal. Statistically significant differences in measured parameters between the control (i.e., untreated) sites and sites grafted with CBBM or CBBM1EGCG were analyzed with one-way ANOVA using SPSS 20 (SPSS, Chicago, IL). The paired t-test was used to compare the parameters describing vertical changes between the buccal and lingual sides in each group. RESULTS

Clinical findings Clinical measurements of the socket dimensions immediately after tooth extraction revealed that the buccolingual ridge was significantly wider for distal sockets treated in the test groups (4.4 6 0.5 mm in the CBBM and 4.3 6 0.5 mm in the CBBM1EGCG groups, respectively) than for the control sockets (3.0 6 0.4 mm) (p < 0.05). However, the thickness of the buccal (0.4 6 0.1 mm in the control, 0.4 6 0.2 mm in the CBBM, and 0.4 6 0.1 mm in the CBBM1EGCG, respectively) and lingual (0.9 6 0.1 mm in the control, 0.8 6 0.2 mm in the CBBM, and 0.9 6 0.1 mm in the CBBM1EGCG, respectively) bone plates did not differ significantly between the three groups. For each extraction socket, the lingual bone plate was significantly thicker than the buccal one (p < 0.05). One dog developed soft-tissue dehiscence and graft material exposure in the CBBM1EGCG-treated site at 1 week after the surgery. The site was resutured and the suture material was removed after 1 week, whereupon uneventful healing occurred. No specific signs of inflammation were found in all sites and samples were available for dimensional measurements (n 5 5). Measurements of dimensional changes in the superimposed images Dimensional changes of the soft-tissue profiles and underlying bone between S0 and S4 were analyzed within the groups. Vertical changes in the soft tissue profiles (VS) were reduced in all groups at the buccal and lingual crevice levels, and the mean value was significantly larger on the buccal side (control with 2.3 6 0.7, CBBM with 2.8 6 0.7, and CBBM1EGCG with 2.5 6 1.0 mm) than on the lingual side (Table I). This reduction did not differ significantly between

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the groups. Soft tissue thickness measured by the vertical distance between S4 and H4 was significantly larger at buccal aspect (control with 3.1 6 0.7, CBBM with 2.7 6 0.6, and CBBM1EGCG with 2.1 6 0.8 mm) compared to lingual side in all groups, but the difference between the groups was insignificant. Figure 2 shows the results of the analysis of the horizontal dimensions of buccal and lingual aspects from the 4mm to the 8-mm level. There were no significant differences in horizontal dimensions between the groups at all levels measured. The difference in the ridge width at the 4-mm level was prominent between the control (1.7 6 2.4 mm) and both test groups (3.3 6 1.7 and 3.0 6 1.7 mm in the CBBM and CBBM 1 EGCG groups, respectively). At 5-mm level, the ridge width was 4.2 6 1.5 mm in control, 4.6 6 1.4 mm in CBBM, and 5.0 6 0.9 mm in CBBM 1 EGCG, respectively. At 6-mm level, the width ranged from 5.5 6 1.3 mm (in control) to 5.9 6 0.9 mm (in CBBM 1 EGCG) and at 7-mm level, the range was 6.4 6 0.8 mm (in CBBM 1 EGCG) to 6.5 6 1.1 mm (in control). Although there was a tendency for greater width at 4-mm level in test groups compared to control group, the differences in the ridge widths between the groups were not statistically significant at all levels. Histologic and histomorphometric analysis In all histologic samples, the extraction socket was filled with NB up to the mesial and distal bone crest of the

FIGURE 2. Horizontal dimensions from the axis of the socket in the buccal and lingual aspects measured at levels of 4–8 mm from the reference line (G0) in sockets without graft material (control) and those treated with CBBM and CBBM1EGCG at 4 weeks postextraction. No statistical differences were shown between the groups in all levels. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.].

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FIGURE 3. Representative photomicrographs of the control group (a) and the two experimental groups: CBBM (b) and CBBM1EGCG (c) in mesiodistal sections at 4 weeks post-extraction. Note that the particles are dispersed in the CTS with limited woven bone in the apical boundaries (arrowheads) in the test groups, while the whole socket is filled with NB in the control group (Masson’s trichrome stain, original magnification of 320).

socket. Some inflammatory cell infiltration and the number of multinucleated cells around RB particles were scattered within the connective tissue space in both experimental groups (Figure 3). A dense, fibrous arrangement of periosteum-like tissue was observed covering the socket entrance of all sites. In the coronal parts, a periosteum-like dense fibrous tissue layer without inflammatory cell infiltration was found in all groups [Figure 4(a–c)]. However, multinucleated cells were found around the RB in both test groups, and limited NB was deposited onto the resorbed surface of RB particles. A similar healing pattern with extensive bone formation was seen in the middle parts [Figure 4(d–f)]. In control sites, a finger-like projection of woven bone had sprouted from the lateral wall of the socket. Conversely, reduced bone formation was found in both test groups. A distinct difference was observed in the apical parts [Figure 4(g–i)]. Especially in the CBBM group, fibrous tissues were observed surrounding the RB at the apical region, separating the biomaterial from the pre-existing adjacent tissues, the lamellar bone, and the marrow bone area. The fibrosis area was smaller in the CBBM1EGCG group than in the CBBM group, and NB was observed within or around the RB in this area. The proportions of NB, RB, and CTS occupying the whole socket are compared between groups in Figure 5(a), and the proportions of the tissue types are compared within the three compartments (coronal, middle, and apical) of the sockets in Figure 5(b). In each test group (CBBM1EGCG and CBBM), one specimen was excluded during measure-

ments due to the large artifact and inappropriate cutting axis during slide preparation. In terms of NB at the apical portion, the control group exhibited the largest area (0.36 6 0.06 mm2; 56.72 6 9.72%), followed by the CBBM1EGCG (0.13 6 0.09 mm2; 20.84 6 14.51%) and CBBM (0.10 6 0.02 mm2; 15.53 6 3.25%) groups. Total area of NB occupying the whole socket in control group also was larger (1.05 6 0.20 mm2; 53.5 6 9.5%) than other two test groups of CBBM1EGCG (0.58 6 0.25 mm2; 27.1 6 13.6%) and CBBM (0.45 6 0.18 mm2; 23.0 6 8.1%). However, comparisons of NB within whole socket or in each part between the groups did not show statistically significant differences. The amount of RB observed in the test groups was similar in all parts of the socket. Both CBBM and CBBM1EGCG sites exhibited marked increases in the number of multinucleated cells around the RB, and NB was deposited onto the resorbed biomaterial surfaces. Multinucleated cells were rarely observed in the control group, with a total count of 37.6 6 14.5, compared to counts of 240.5 6 62.8 and 197.7 6 51.4 in the CBBM and CBBM1EGCG sites, respectively. The cell count was significantly lower in the control group than in the two test groups, and did not differ significantly between the two test groups.

DISCUSSION

Information about the healing process in relation with the presence of periapical lesion and the response of grafted materials to the infection sources within the socket is limited. A systematic review has described that the placement

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FIGURE 4. Higher magnification photomicrographs of the boxes outlined in Figure 3. The NB around the periapical lesion in the CBBM site is clearly separated by a dense fibrous layer (arrows), while woven bone is evident within the internal pore and on the resorbed surface of biomaterials at the CBBM1EGCG site (Masson’s trichrome stain, original magnification of 3100). Arrowheads, multinucleated cells; asterisks, RB particles. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.].

of implants into sites with periapical and periodontal infections might be an acceptable procedure when proper protocol of thorough mechanical debridement was preceded.4

However, some human studies have reported controversial results about the compromised osseointegration, retrograde peri-implantitis, and increased risk of implant failure in the

FIGURE 5. The proportions of different tissues in the entire socket (a) and in the apical, middle, and coronal compartments of the socket (b). A, apical; M, middle; C, coronal. No statistical differences were shown between the groups in %NB, %RB, and %CT within the entire socket and in each part (A, M, and C). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.].

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sites formerly infected even after a long time of healing.16– We have used a model of induced periapical lesion from pulpal necrosis to focus on the response of grafting material in an infected socket and the effect of an anti-inflammatory agent, EGCG, on the healing process. For the purpose of this study, the lesions were not debrided to assure that the infection source remained. This extreme condition of an uncontrolled infection was intended to maximize the difference in healing patterns between the grafted and nongrafted sockets in relation to the inflammatory response. Superimposition of digital images taken from laserscanned cast models (S0 and S4) and micro-CT scans (H4) together was used to visualize changes in soft tissue levels over the final hard tissue profile of the infected sockets during 4 weeks of healing. It enabled the measurement of both soft and hard tissue dimensions at any location when appropriate reference points were set, compared to the previous studies mostly demonstrating soft tissue changes just using dental cast model alone in both human and other animals.7,10,27 However, there were some limitations in the data that comparison in the hard tissue change between two time points was missing as the micro-CT image before surgery was not taken and only the vertical change in the soft-tissue profile was shown due to the inconsistent folding of the vestibular area that occurred during taking the impressions. Dimensional changes in soft tissue during 4 weeks of healing were similar between the groups to show significant reduction in buccal aspects, whether the infected socket was immediately grafted or healed naturally. Although the longitudinal data for soft tissue alone were limited, the alteration appeared to be substantial in combination with hard tissue, which was similar to the pattern of underlying bone resorption.28,29 In general, the outline of soft tissue appeared to reflect the contour of underlying bone, and the vertical discrepancy could represent the extent of buccal bone resorption consistently found in all the groups. Horizontal dimension in hard tissue at 4 weeks showed similar pattern with no significant difference between the groups and had pronounced bone loss in buccal aspect. The baseline thickness of the bone plates in the buccal or lingual sides measured immediately after tooth extraction was similar in the mesial and distal sockets. It is known that any change in buccolingual dimension is due mainly to resorption of the buccal bone plate,30 and so the difference in the baseline ridge width between the sockets would not be a critical bias in this experimental study. Previous studies of dog models with immediate grafting in fresh extraction sockets have described the maintenance of hard tissue volume in coronal part of alveolar ridge.12,31 It has been suggested that grafting materials counteract the alteration of modeling and remodeling during the natural healing process that is responsible for buccolingual contraction of alveolar ridge after tooth extraction. In this study, when the infected socket was grafted with CBBM alone or CBBM with EGCG, neither was involved in more destructive bone loss nor significantly preserved the contour of alveolar ridge compared to the control group. However, there was a tendency toward increased bone loss in the buccal aspect of the non-grafted site 19

within the coronal 4-mm level examined from the gingival margin, where the position of implant shoulder would take place in future installation. It is possible that the influence of periapical lesion might have only limitedly reached the upper part of the socket to hinder the healing process related to the biomaterials and newly formed bone. To clarify the tendency in wider coronal width of grafted groups, further study with larger sample size is needed. Histomorphometric measurement of %NB in entire or in each part of the socket was greater in the control group compared to the test groups and the difference in NB formation seemed to be more prominent at the apical area, although statistically insignificant due to the small sample size. Histologic observation in control group also followed the natural healing process in fresh extraction socket,12,13 which usually appeared to have new bone formation started from the apical and lateral walls and extended to the coronal and center portion. However, the healing of infected sockets treated with graft materials revealed some unique features at the apical regions of both test groups, which exhibited limited bone formation and some localized areas of densely arranged fibrous tissues (fibrosis) with dispersed particles of biomaterial in the CTS and scarce findings of cellular entities. Although the quantitative measurement in %NB was insignificant between CBBM with or without EGCG, remaining particles of CBBM group in apical region were mostly associated with the multinucleated cells on the surface, whereas direct contact of NB on the particle surface was limitedly found in CBBM with EGCG group. It is possible that the granulation tissue found in the presence of a periapical lesion resulted in the healing by a reparative process rather than bone regeneration during a foreign-body reaction with the xenograft and the adjunctive use of EGCG might partly suppress the inflammatory response. There have been too few controlled experimental studies to allow a reliable comparison of the incidence, extent, and effects of retrograde infection; however, some case reports and retrospective studies on retrograde peri-implantitis have described long duration of localized resident infection in most apical part.19,20 In vitro and in vivo studies have demonstrated the effects of EGCG in the depression of inflammation and tissue destruction related to osteoclastic activity.23–25,32–34 There was a tendency toward a decreased cell count in the CBBM with EGCG group, which suggested the involvement of anti-inflammatory effects that would also reduce the degree of fibrosis and increase the bone formation in the healing socket. As a pilot study with limited sample sizes and histologic specimen, the quantitative results could not be clearly demonstrated with statistical significance. Still, there was a tendency toward the maintenance or reduced buccal dimensional loss in the coronal aspect of the infected socket with CBBM, but the apical region of the socket seemed to be under consistent influence of the periapical lesion to show reparative healing after 4 weeks of healing. In limitations to the qualitative data, it also could be suggested that the use of EGCG had beneficial effect on the control of the inflammatory response to CBBM and reduction in the extent

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of fibrosis at the apical area. Further studies are needed to quantify the effects of EGCG, mechanisms of the biologic activity and to find out the appropriate concentration and delivery systems controlling the release of EGCG. Within the limitations of this study, it was conjectured that adjunctive use of EGCG with CBBM can be a candidate biomaterial in grafting of extraction socket with periapical lesion, despite its acute infection state. Bone regeneration at the coronal region of the CBBM grafted socket might not be influenced by the presence of a periapical lesion. The role of fibrosis in socket healing and the possibility that the fibrotic tissue could be a source of reinfection should be closely examined in further long-term studies involving larger samples. REFERENCES 1. Lin SK, Kok SH, Kuo MY, Wang TJ, Wang JT, Yeh FT, Hsiao M, Lan WH, Hong CY. Sequential expressions of MMP-1, TIMP-1, IL6, and COX-2 genes in induced periapical lesions in rats. Eur J Oral Sci 2002;110:246–253. 2. Wang CY, Tani-Ishii N, Stashenko P. Bone-resorptive cytokine gene expression in periapical lesions in the rat. Oral Microbiol Immunol 1997;12:65–71. 3. Vier FV, Figueiredo JA. Prevalence of different periapical lesions associated with human teeth and their correlation with the presence and extension of apical external root resorption. Int Endod J 2002;35:710–719. 4. Waasdorp JA, Evian CI, Mandracchia M. Immediate placement of implants into infected sites: A systematic review of the literature. J Periodontol 2010;81:801–808. 5. Quirynen M, Gijbels F, Jacobs R. An infected jawbone site compromising successful osseointegration. Periodontol 2000 2003;33: 129–144. 6. Lekovic V, Kenney EB, Weinlaender M, Han T, Klokkevold P, Nedic M, Orsini M. A bone regenerative approach to alveolar ridge maintenance following tooth extraction. Report of 10 cases. J Periodontol 1997;68:563–570. 7. Tan WL, Wong TL, Wong MC, Lang NP. A systematic review of post-extractional alveolar hard and soft tissue dimensional changes in humans. Clin Oral Implants Res 2012;23 Suppl 5:1–21. 8. Botticelli D, Berglundh T, Lindhe J. Hard-tissue alterations following immediate implant placement in extraction sites. J Clin Periodontol 2004;31:820–828. 9. Lang NP, Pun L, Lau KY, Li KY, Wong MC. A systematic review on survival and success rates of implants placed immediately into fresh extraction sockets after at least 1 year. Clin Oral Implants Res 2012;23 Suppl 5:39–66. 10. Vignoletti F, Matesanz P, Rodrigo D, Figuero E, Martin C, Sanz M. Surgical protocols for ridge preservation after tooth extraction. A systematic review. Clin Oral Implants Res 2012;23 Suppl 5:22–38. 11. Araujo M, Linder E, Lindhe J. Effect of a xenograft on early bone formation in extraction sockets: An experimental study in dog. Clin Oral Implants Res 2009;20:1–6. 12. Araujo M, Linder E, Wennstrom J, Lindhe J. The influence of BioOss Collagen on healing of an extraction socket: An experimental study in the dog. Int J Periodontics Restorative Dent 2008;28:123– 135. 13. Araujo MG, Liljenberg B, Lindhe J. Dynamics of bio-oss collagen incorporation in fresh extraction wounds: An experimental study in the dog. Clin Oral Implants Res 2010;21:55–64. 14. Del Fabbro M, Boggian C, Taschieri S. Immediate implant placement into fresh extraction sites with chronic periapical pathologic features combined with plasma rich in growth factors: Preliminary results of single-cohort study. J Oral Maxillofac Surg 2009; 67:2476–2484. 15. Siegenthaler DW, Jung RE, Holderegger C, Roos M, Hammerle CH. Replacement of teeth exhibiting periapical pathology by immediate implants: A prospective, controlled clinical trial. Clin Oral Implants Res 2007;18:727–737.

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EFFECTS OF EGCG ON RIDGE PRESERVATION

Effects of epigallocatechin-3-gallate on the healing of extraction sockets with a periapical lesion: A pilot study in dogs.

The aim of this study was to characterize the healing process of extraction sockets with a periapical lesion following immediate graft with collagenat...
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