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Title

Periodontal regeneration following transplantation

of proliferating tissue derived from periodontal

ligament into class III furcation defects in dogs

Author(s)

Alternative

Murano, Y; Ota, M; Katayama, A; Sugito, H;

Shibukawa, Y; Yamada, S

Journal Biomedical research, 27(3): 139-147

URL http://hdl.handle.net/10130/52

Right

Biomedical Research 27 (3) 139-147, 2006

Periodontal regeneration following transplantation of proliferating tissue de-rived from periodontal ligament into class III furcation defects in dogs

Yoshinori MURANO, Mikio OTA, Akihiko KATAYAMA, Hiroki SUGITO, Yoshihiro SHIBUKAWA and Satoru YAMADADepartment of Periodontology, Tokyo Dental College, Chiba, Japan

(Received 6 March 2006; and accepted 25 April 2006)

ABSTRACTThe aim of this study was to evaluate the healing of class III furcation defects following trans-plantation of proliferating tissue derived from periodontal ligament (pPDL). Two weeks after re-moving alveolar bone, pPDL was excised. Class III furcation defects were created in the mandibular premolars. pPDL was transplanted into the furcation defects in the experimental group, while no treatment was performed on the furcation defects in the controls. Two, four and eight weeks after surgery, histologic examination, quantitative RT-PCR, and immunohistochemistry were carried out. bFGF and VEGF mRNA showed a significant increase in pPDL. In the pPDL treat-ment group, new cementum regenerated around almost the entire circumference of the furcation, with new bone filling most of the defect, while the control group presented epithelial downgrowth and defects filled with connective tissue. These results provide histological evidence that pPDL plays an important role in wound healing by promoting periodontal regeneration in class III furca-tion defects.

Periodontal regeneration aims at the restitution of supporting periodontal tissues lost due to periodon-tal diseases (2). The effectiveness of various growth factors has been demonstrated by studies using ani-mals on periodontal regeneration (3, 14). Guided tis-sue regeneration (GTR) therapy has been reported to achieve repopulation of periodontal ligament (PDL) fibroblasts and to promote periodontal regen-eration in animals and humans (7, 9). Sugimoto et al. (26) investigated whether proliferating tissue de-rived from periodontal ligament (pPDL) in peri-odontal osseous defects could form new periodontal ligament. Immunohistochemical studies of pPDL re-vealed the presence of a number of matrix macro-molecules such as collagen types I, III, and IV, vitronectin, fibronectin, bone sialoprotein, and osteo-

nectin (1, 13). This discovery was consistent with the findings reported by Ivanovski et al. (12). They reported that mRNA expression of alkaline phospha-tase, osteonectin, bone sialoprotein, and osteopontin increased in pPDL (12). The results of those studies suggest that periodontal regeneration is achieved through pPDL because of its abundant matrix com-ponents specific to distinct periodontal components. pPDL applied to the surface of dentin block im-plants has recently been investigated by in vivo study (26). Over an 8-week observation period, the implanted root bearing pPDL formed new PDL on the surface. Therefore, the authors concluded that pPDL promoted new PDL formation around the root (26). Lang et al. (16) reported similar results in dogs. These studies indicated that various cells mi-grated from pPDL which were basically capable of synthesizing periodontal tissue after transplantation.　The classification of the furcation defect is based on the amount of periodontal tissue; class I: hori-zontal loss of periodontal support not exceeding 1/3 of the width of the tooth, class II: horizontal loss of

Address correspondence to: Dr. Yoshinori Murano, De-partment of Periodontology, Tokyo Dental College, 1-2-2 Masago, Mihama-ku, Chiba 261-8502, JapanTel: +81-43-270-3954, Fax: +81-43-270-3955E-mail: ymurano@tdc.ac.jp

Y. Murano et al.140

experiment day 0, a re-entry surgical procedure was performed on the mandibular 1P1 to remove the membranes and obtain the pPDL, which was then excised. In this study, pPDL was used for transplan-tation. The harvested tissue was kept moist in sterile saline until used. Part of the pPDL was also used to examine mRNA expression of basic fibroblast growth factor (bFGF) and vascular endothelial cell growth factor (VEGF) using a LightCycler™ (F. Hoffmann-La Roche Ltd, Basel, Switzerland).

Surgical procedure. Five weeks before experiment day 0, class III furcation defects were created surgi-cally at the third and fourth mandibular premolars using bone chisels and slow-rotation diamond burs. The alveolar bone of each premolar (3P3, 4P4) was also removed, creating a “horizontal” pattern of bone loss (Fig. 1). The furcation defects were ap-proximately 4 mm wide and 4 mm high and were filled with impression material (Exafine®, G.C. Corp. Tokyo, Japan). The flaps were repositioned and sta-bilized with silk sutures. The impression material was removed from the defects 2 weeks before ex-periment day 0. At experiment day 0, after elevation of the mucoperiosteal flaps of the class III furcation defects, granulation tissue was removed and the root surface was thoroughly debrided using curets. A notch was placed on either side of the root at the level of the reduced bone crest. During this time, the pPDL was retrieved as described above. On one side of the class III furcation defects for the mandi-ble premolars (experimental groups), pPDL was transplanted into the horizontal furcation defects. The contralateral premolars (control group) received the same treatment, but pPDL was not applied. Mu-coperiosteal flaps were raised and sutured in a coro-nal position.

periodontal support exceeding 1/3 of the width of the tooth, and class III: horizontal “through and through” destruction of the periodontal tissues in the furcation area. Various surgical procedures have been developed for healing class II or III furcation defects (4, 6), and various bone grafts and alloplas-tic materials have been transplanted (5, 24, 29); however, ankylosis, resorption, and long epithelial attachment are observed in alloplastic material-ap-plied cases (8). It has been demonstrated that GTR is capable of successfully closing class III furcation defects in a dog model (11, 17, 18); however, the regeneration of class III furcation defects is often incomplete, even when GTR is employed, suggest-ing that this treatment is not effective for large, class III furcation defects (15, 21, 22), as it depends on natural healing ability. Since pPDL is basically capable of synthesizing periodontal tissue after transplantation, this treatment may be applicable to large periodontal tissue defects. Therefore, applica-tion of pPDL to class III furcation defects offers on attractive proposition.　The objective of this study was to investigate the effect of pPDL on periodontal tissue formed after transplantation in class III furcation defects in dogs.

MATERIALS AND METHODS

Fifteen healthy mongrel dogs were used for this study. The animals were placed under general anes-thesia with ketamine hydrochloride (KETALAR®50; SANKYO, Tokyo, Japan) at a dosage of 10 mg/kg. In order to reduce haemorrhagia in surgical areas, a local infiltration anesthesia (2% Xylocain®, 1 : 80,000 epinephrine; Astellas, Tokyo, Japan) was also used. The second mandibular premolars (2P2) were ex-tracted for pPDL as described below. All experi-ments were performed according to the Guidelines for the Treatment of Experimental Animals at Tokyo Dental College.

Preparation of pPDL. Three months after the ex-tractions of 2P2, at 2 weeks before the experiment commenced (day 0), periodontal osseous defects were created around mandibular 1P1 to induce tissue growth under the membrane (hereafter referred to as pPDL). This method for induction of pPDL has been described previously by Sugimoto et al. (26). The root surface was then thoroughly scaled and planed. The defect was next covered with an e-PT-FE membrane (GTAM Oval-9; WL Gore & Associ-ates, Flagstaff, AZ, USA). The flaps were then replaced and sutured. Two weeks after surgery at

Fig. 1　Surgically induced class III furcation defect at the third and fourth mandibular premolars (asterisk).

Periodontal regeneration of furcation defect by pPDL 141

analysis of variance and the multiple comparison Scheffé test were used to analyze the data.

Preparation of samples for quantitative RT-PCR us-ing LightCycler™. A re-entry surgical procedure was performed to obtain biopsies of pPDL (the pro-liferating tissue group) during the 2-week period af-ter the surgery described above. Samples of the connective tissues of the gingiva (the connective tis-sue group) were separated from other gingival tis-sues. Total RNA was extracted from these samples by the acid guanidine thiocyanate/phenol-chloroform method. The samples were excised, then homoge-nized and solubilized in TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and chloroform. Supernatants were obtained by centrifugation at 13 200 rpm for 20 min at 4°C, added to isopropanol, stored for over 1 h at −80°C, and finally centrifuged at 13 200 rpm for 20 min at 4°C. The precipitates were obtained by decantation, and washed with 70% ethanol. The RNA pellets were dissolved in RNase-free water, and kept at −80°C until used. Total RNA was mea-sured by absorbance in an UVmini-1240 (Shimadzu Corporation, Kyoto, Japan). Oligo dT primer: 1 μL, dNTP: 2 μL, RNase inhibitor: 1 μL, reverse tran-scriptase: 1 μL, 10 × buffer: 2 μL, MgCl2: 4 μL were added to a total of 1 μg of RNA, and the total vol-ume was adjusted to 20 μL with RNase-free water. The mixture was then reverse transcripted (42°C for 15 min, 99°C for 5 min) to synthesize cDNA. PCR was then carried out using primers for bFGF, VEGF, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Table 1). To measure mRNA levels, quantitative PCR assays were conducted with a LightCycler™ using double-stranded DNA dye SYBR Green I (Roche Diagnostics, Manheim, Ger-many), and quantification was performed by com-paring the levels obtained to standardized samples. The PCR conditions used in the LightCycler™ were: 45 cycles bFGF (95°C for 10 sec, 62°C for 10 sec, 72°C for 6 sec); 45 cycles VEGF (95°C for 10 sec, 57°C for 5 sec and 72°C for 8 sec); 45 cy-

Histological processing. The animals were eutha-nized with an intravenous overdose of sodium pen-tobarbital at two, four, and eight weeks following the procedure described above. The jaw of each ani-mal was then removed, and specimens containing the experimental areas were placed in 20% buffered formalin. Specimens were decalcified with 10% eth-ylenediaminetetra acetic acid (EDTA; Wako, Tokyo, Japan) for 5 months and then dehydrated in ethanol, embedded in paraffin, and serially sectioned (to 4 μm thickness) in the mesio-distal orientation. The sections were then stained with hematoxylin-eosin. Three sections, 100 μm apart, representing the cen-tral area of each furcation, were selected from each tooth for linear measurements. In each section, the following linear distances were assessed: (1) the dis-tance from the line marking the apical border be-tween the notches on the mesial and distal roots to the fornix of the furcation, i.e., the height of the de-fect; (2) the distance from the line marking the api-cal border of the notches on the mesial and distal roots to the most coronal level of the newly formed alveolar bone, i.e., the height of the newly formed bone; (3) the circumference of the defect between the apical borders demarked by the notches on the mesial and distal roots; and (4) the total distance along the apical borders from the notches to the cor-onal level of newly formed cementum on the mesial and distal roots, i.e., the amount of new cementum. All distances were measured in millimeters (mm). The newly formed bone was also expressed as a percentage (%) of the height of the defect and the amount of new cementum as a percentage of the to-tal circumference of the defect. Measurements were made using the following image analysis system: a light microscope (Olympus BX51 Microscope, Olympus Optical Co., Tokyo, Japan) with a × 4 ob-jective, equipped with a digital camera (HC-500, FUJI FILM, Tokyo, Japan) coupled to a computer (DELL, Precision Work Section 220) with software used for image processing (Image Pro Plus v 3.0 Media Cybernetics, Silver Spring, MD, USA). An

Table 1　Primer sequences and product sizes

Gene Sequence Product size

bFGF5’-CAATACTTACCGGTCAAGG-3’

103bp5’-TATAGCTTTCTGCCCAGGT-3’

VEGF5’-AGGAGTTCAACATCACCAT-3’

127bp5’-TCTTGCCTTGCTCTATCTT-3’

GAPDH5’-TGGGAAGATGTGGCGTGAC-3’

168bp5’-CGGCAGGTCAGATCCACAACT-3’

Y. Murano et al.142

cles GAPDH (95°C for 10 sec, 50°C for 5 sec, 72°C for 8 sec). Melting curve analysis was performed af-ter PCR amplification to confirm the absence of the primer dimer in the PCR products. The ratios of bFGF and VEGF mRNAs were corrected for the value of the housekeeping gene GAPDH. The PCR products were separated on 2% agarose ethidium bromide gels. Each PCR fragment was verified as canis bFGF: AF060562, canis VEGF: AF149417 or canis GAPDH: AB038240 (Nihon Gene Research Labs Inc., Tokyo, Japan). The Scheffé test was used for statistical analysis.

Immunohistochemistry for PCNA. We carried out the following immunohistochemical analysis with mouse anti-proliferating cell nuclear antigen (PCNA) pri-mary antibody. Paraffin sections (approximately 4 μm thick) were cut, and immunohistochemical staining of PCNA was performed using an immuno-peroxidase staining kit (Histofine SAB-PO (M) kits; NICHIREI, Tokyo, Japan). The sections were incu-bated with mouse anti-PCNA primary antibody (PC-10; DAKO Corporation Carpinteria, CA, USA) at a dilution of 1 : 100. Next, each section was incu-bated with biotinylated secondary antibody and streptavidin peroxidase reagents. The presence of peroxidase-complexes was visualized by 3-3’ diami-nobenzidine tetrahydrochloride (0.1 mg/mL) solution with 0.65% H2O2. Sections were counterstained with Mayer’s hematoxylin. Magnification was set at × 400. A field of connective tissue from immediately beneath the coronal to the apical area of the furca-tion defect was selected in each section, and a 0.12 mm2 (0.2 mm × 0.6 mm) area was submitted to quantitative analysis. The number of PCNA-positive cells in the furcation area was calculated. An analy-sis of variance and the multiple comparison Scheffé test were used to analyze the data.

RESULTS

Two weeks after surgeryIn experimental group, the furcation lesion was oc-cupied by slightly inflamed and newly formed con-nective tissue (Fig. 2A). The slightly inflamed connective tissues that occupied the coronal half of the lesion were composed of many fibroblasts and collagen fibers, a small number of inflammatory cells, and primary lymphocytes adjacent to small blood vessels (Fig. 2B). The newly formed connec-tive tissue (asterisk in Fig. 2A), which extended into the interradicular area, was distinguished from the slightly inflamed tissue by the presence of many

blood vessels, abundant collagen fibers, and numer-ous well-polarized fibroblasts (Fig. 2B). In control group (data not shown), the most coronal portion of the lesion contained bacterial plaques on the root surface. The furcation defect was occupied by in-flammatory connective tissue covered by gingival epithelium. The middle portion of the connective tissue contained more inflammatory cells, and the apical portion was composed of new fibrous connec-tive tissue.

Four weeks after surgeryIn experimental group, the furcation lesion was completely healed, filling with new fibrous connec-tive tissue and bone (Fig. 2C). The coronal half was filled with connective tissue, whereas the apical por-tion had formed new bone on the alveolar bone. The fornix root surface of the furcation was covered with thick collagen fibers. Active bone formation was observed in the interradicular area (Fig. 2D). In control group (data not shown), the coronal half of the lesion was occupied by the gingival epithelium and slightly inflamed connective tissue beneath the gingival epithelium. The connective tissue was com-posed of fibroblasts and a small number of inflam-matory cells. The apical portion was repaired and formed a small amount of new bone.

Eight weeks after surgeryIn experimental group, the lesion had almost regen-erated, with new cementum along the root surface, new bone and new PDL (Fig. 2E). The root surface of the furcation area was almost completely covered with new cementum, and Sharpey’s fibers inserting into the new cementum were observed (Fig. 2F); however, complete alveolar bone reconstruction was not achieved. Bone ankylosis and root resorption were not observed on the root surface. In control group, all furcations were open, showing inflamed connective tissue covered by gingival epithelium in the lower portion of the defect, and new cementum with inserted collagen fibers and new bone were limited to the level of the notch (Fig. 2G).

Immunohistochemistry for PCNAThe staining results for PCNA at week 4 after sur-gery in the newly formed connective tissue are shown in Fig. 3A. The experimental group showed more PCNA-positive fibroblast-like cells than the control group (Fig. 3B). Results indicating the num-ber of PCNA-positive cells in the furcation area are summarized in Table 2. PCNA-positive cells in the experimental group were significantly greater than

Periodontal regeneration of furcation defect by pPDL 143

in the control group (36.3 ± 2.5 vs 10.9 ± 2.1: p < 0.01).

Histomorphometric MeasurementEfficacy of treatment in the experimental group was evaluated by histomorphometric analysis of furca-tion defect regeneration at 8 weeks after surgery. The results are summarized in Table 3. The experi-mental group showed accelerated periodontal regen-eration with new cementum by 98.1 ± 2.3%, and new bone by 84.8 ± 3.9%. In contrast, new cemen-tum and new bone in the control group were 22.3 ± 1.6% and 12.2 ± 1.3%, respectively. This demon-strated that the amounts of new cementum and bone in the experimental group were significantly higher (p < 0.01) than in the control group.

mRNA expression analyses of proliferating tissuesExpression of bFGF and VEGF mRNA was investi-gated at week 2 after surgery with RT-PCR using a LightCycler™. This revealed that bFGF mRNA ex-pression was detectable in the majority of samples from the pPDL group. It also showed that mRNA expression of bFGF and VEGF was higher in the pPDL group than in the connective tissue group (Fig. 4). The bFGF mRNA levels were significantly higher in the pPDL group than in the connective tissue group (p < 0.01) (Fig. 4B). VEGF mRNA expression was detected at significantly higher lev-els in the pPDL group than in the connective tissue group (p < 0.01) (Fig. 4C).

Fig. 2　Histology of periodontal healing of class III furcation defects (H&E staining). Squares indicate an area of the higher magnification. A~F: Experimental group. A, B: Two weeks after surgery, the coronal portion (CP) of a furcation is occupied by the slightly inflamed connective tissue, while the apical portion contains fibrous connective tissue (asterisk). B: Note nu-merous fibroblasts and many small blood vessels (arrowheads) in the fibrous connective tissue. (original magnification A: 20, B: × 160) C, D: Four weeks after surgery, a furcation of the defect contains fibrous connective tissue and new bone (NB). AB alveolar bone (original magnification C: × 6.25, D: × 80) E, F: Eight weeks after surgery, a regeneration furcation is filled with new bone (NB), PDL (asterisks) and cementum (arrowheads). AB alveolar bone F: Note newly formed cementum (NC), bone (arrowheads), and regenerated periodontal ligament. (original magnification E: × 6.25, F: × 80) G, H: The control. Eight weeks after surgery, new bone (NB) has formed in the notch area. The remaining part of the furcation area presents in-flamed connective tissue (asterisk) covered with epithelium (arrowheads). (original magnification G: × 6.25, H: × 80)

Y. Murano et al.144

sequence of events accompanied by the formation of new cementum, PDL, and bone.　We found that local application of pPDL to class III furcation defects significantly increased peri-odontal regeneration without ankylosis or epithelial downgrowth. We also confirmed that local applica-tion of pPDL to class III furcation defects accelerat-

DISCUSSION

We investigated whether local application of pPDL to class III furcation defects could effectively induce periodontal regeneration. It was demonstrated that pPDL treatment successfully healed class III furca-tion defects, and that the healing process involved a

Table 2　Number of PCNA positive cells in connective tissue of furcation defect

Experimental group(mean ± SD)

Control group(mean ± SD)

PCNA-positive cells 36.3 ± 2.5 10.9 ± 2.1

* Stastistically significant: p < 0.01 (n = 5)

*

Table 3　Histometric parameters of each treatment

Experimental groupmean ± SD (%)

Control groupmean ± SD (%)

new cementum 98.1 ± 2.3 22.3 ± 1.6

new bone 84.8 ± 3.9 12.2 ± 1.3

* Stastistically significant: p < 0.01 (n = 5)

**

Fig. 3　Immunohistochemistry for PCNA in experimental group (A) and control group (B) at 4 weeks after surgery. PCNA-positive cell numbers at experimental group (A) are significantly greater than at control group (B). Sections were stained with streptavidin-biotinylated antibody (SAB) and counterstained with Mayer’s hematoxylin at 4 weeks after surgery. (Original magnification × 100)

Periodontal regeneration of furcation defect by pPDL 145

by RT-PCR. The VEGF mRNA level increased in the pPDL compared with connective tissue (control group). This study showed that growing capillaries associated with abundant fibroblasts (mesenchymal cells) invaded the wound region from surrounding bone marrow 2 weeks after surgery in the pPDL treatment group. These findings demonstrate that pPDL with a high VEGF expression level supplies abundant blood vessels to the wound. Several growth factors, such as VEGF and bone morphoge-netic proteins, modulate bone formation and angio-genesis during bone wound healing. Previous studies have demonstrated that immunoreactivity for VEGF can be detected in chondrocytes and osteoblasts in the fracture callus during healing process (23, 28). A recent study demonstrated that inhibition of VEGF decreased angiogenesis, bone formation, and callus mineralization in a mouse bone injury model (25). It has been shown that VEGF is essential in the coordination of angiogenesis and bone forma-tion, and that it may play a crucial role in bone for-mation and neovascularization. Therefore, these results suggest that pPDL with a high VEGF expres-sion level plays a role in the bone tissue regenera-tion of periodontal tissue in furcation defects through promotion of calcification.　Furthermore, our study detected an increase in bFGF mRNA expression level in pPDL. The bFGF is known to promote the proliferation of mesenchy-mal cells, including osteocytes and PDL cells, and to stimulate the osteogenic expression of bone mar-row stromal cells (19). It has been reported that bFGF showed mitogenic activity on undifferentiated

ed periodontal regeneration. Periodontal regeneration in the region to which pPDL was applied was ac-companied by new cementum in 98.1 ± 2.3% of the circumference of the defect, and new bone in 84.8 ± 3.9% of the height of the defect. In the control re-gion, in contrast, regeneration was accompanied by new cementum in 22.3 ± 1.6% of the circumference of the defect and new bone in 12.2 ± 1.3% of the height of the defect. These findings show that the wound healing process induced by pPDL treatment is important for new cementum and new bone for-mation. At 2 weeks after regeneration therapy using pPDL, the furcation defect was composed of fibrous connective tissue. At 4 weeks, the entire furcation was filled with fibrous connective tissue and this tissue at 8 weeks was replaced by new bone, PDL, and cementum. No inflammation or edema was noted in any region in the 2 weeks group of the pPDL treatment group. The furcation area was not exposed, following pPDL application during this pe-riod. Incomplete regeneration of the furcation defect in the control group was due to insufficient wound closure. These findings suggest that application of pPDL promotes cells population of the bone and root surfaces of furcation defects, and that periodon-tal regeneration requires a certain time period to take place. Epithelial downgrowth was noted in the furcation defect at 2 weeks after surgery in the con-trol group. The early appearance of stable tissue on the root surface interferes with epithelial growth to-ward the root surface, as clearly seen in the pPDL treatment group (16).　VEGF expression level in pPDL was determined

Fig. 4　mRNA expression of bFGF, VEGF and GAPDH by LightCyclerTM. A: RT-PCR analysis. Observed single band indi-cates that each primer was specific to bFGF and VEGF. B and C: Ratio of expression levels in pPDL and connective tissue (CT) group. bFGF and VEGF mRNA expressions were greater in pPDL group than in connective tissue group.

Y. Murano et al.146

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mesenchymal cells, and that undifferentiated mesen-chymal cells proliferated upon stimulation with bFGF (27). In our study, there were more PCNA-positive cells around the new bone tissue in the fur-cation defects in the pPDL treatment group than in the control group, suggesting that bFGF in the pPDL induced the proliferation of undifferentiated mesenchymal cells which then differentiated into osteoblasts and cementoblasts. Therefore, bFGF may increase the potential for desirable periodontal re-generation, mainly bone regeneration, in the furca-tion area. This agrees with the findings of an earlier study by Nakahara et al. (20).　The regeneration of both hard and soft tissues is essential for complete periodontal regeneration. pPDL affects several aspects of periodontal regener-ation such as osteogenesis, cementogenesis, and connective tissue regeneration. None of these pro-cesses was noted in the control group. These results support the findings of our previous study showing that treatment without pPDL inhibited periodontal wound healing (12). In the present study, epithelial downgrowth occurred in the furcation defect in the control group. In contrast, pPDL treatment effective-ly stimulated periodontal regeneration within a short period. Therefore, this study proposes a novel thera-peutic approach and a new research strategy for dental and craniofacial surgery.　In conclusion, this study found histological evi-dence that treatment with pPDL induced periodontal regeneration in mandibular class III furcation de-fects. This suggests that the healing pattern induced by this treatment is more advantageous than that of flap surgery alone.

Acknowledgements

The authors wish to express our sincere gratitude to all members of the Department of Periodontology, Tokyo Dental College for their continuing guidance and encouragement.

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