Lymphatic Architecture of the Human Gingival Interdental Papilla

146 Lymphology 44 (2011) 146-154 LYMPHATIC ARCHITECTURE OF THE HUMAN GINGIVAL INTERDENTAL PAPILLA Y. Ando, O. Murai, Y. Kuwajima, S. Furukawa, D. Sasaki, Y. Okawa, T. Yaegashi, H. Miura, A. Fujimura Department of Anatomy (YA,AF), Division of Functional Morphology, Iwate Medical University; Department of Oral Function Preservation (OM,DS,YO,TY), Division of Periodontics and Endodontics, School of Dentistry, Iwate Medical University; and Department of Developmental Oral Health Science (YK,SF,HM), Division of Orthodontics, School of Dentistry, Iwate Medical University, Iwate, Japan ABSTRACT This may be an important anatomical feature during inflammation throughout the oral Many studies have investigated the cavity in that the drainage function is lymphatic architecture of head and neck using maintained by part of lymphatic flow that is experimental animals, confirming the not impaired during the healing process. existence of lymphatic networks beneath the epithelium in gingival tissue. In this study, we Keywords: lymphatic architecture, investigated the use of these lymphatics as a interdental papilla, human gingiva, drug delivery route by studying the 5’-nucleotidase, D2-40 architecture of lymphatic vessels in human interdental papilla. Serial cryosections were The majority of lymphatic vessel research cut using the film-transfer method. To identify in periodontal tissues has involved the use of lymphatics, the sections were stained using experimental animals (1-8), and the existence enzyme histochemical and immunohisto- of lymphatic networks beneath the epithelium chemical techniques and three-dimensional in gingival tissues has been confirmed. Some images of lymphatics were reconstructed using investigators have suggested that these 3D visualization software. Capillary lymphatic networks could be utilized for clinical treat- networks were observed in the lamina propria ment (4,7,8). Generally, gingival tissues are beneath the epithelium in human interdental divided into the free gingiva, the attached papilla, and they joined with lymphatic gingiva, and the interdental papilla (gingival networks beneath the epithelium in free tissue between the teeth). The tip of the gingiva. The networks consisted of a single pyramid-shaped interdental papilla is layer of large irregular, hexagonal meshes and positioned immediately below the proximal precollecting lymphatic vessels heading toward contacts, and the gingival col, a V-shaped collecting lymphatic vessels that exited on the depression connecting the buccal and lingual periosteum of the alveolar crest. These interdental papillae, is adapted to the shape findings suggest that lymphatic flow from the of proximal contact area of the tooth (9-11). interdental papilla drains into collecting This area has a suitable shape for retention of lymphatic vessels running buccolingually on ointment medicine in dental treatment. the alveolar crest of the interdental papilla. Moreover, the attached epithelium, the Permission granted for single print for individual use. Reproduction not permitted without permission of Journal LYMPHOLOGY. 147 bottom of the gingival sulcus, has a high clinically healthy gingiva as the area located substance permeability because of the wide near the normal tissues without severe intercellular space (12). Medication entering inflammation. The incised gingival tissue was into the lamina propria just beneath the cryo-embedded in 5% carboxymethyl epithelium is absorbed by the blood and cellulose (Kanto Chemical Co., Inc., Tokyo, lymphatic vessels. A recent study summarized Japan) in hexane cooled by liquid nitrogen the existence of lymphatic vessels in various without fixation. The specimen was then human organs (13), but few studies have placed in a cryostat (CM3050S, Leica, investigated the distribution of lymphatic Bensheim, Germany: cutting edge angle: 5°, vessels in the head and neck region focusing CT: -22°C, OT: -18°C) and 10 µm bucco- on the separate gingival tissues. Although we lingual or mesiodistal serial cryosections were have been investigating human lymphatic produced using the film-transfer (Kawamoto) architecture in the buccal and lingual free method (15). gingiva (14), no studies have conducted a detailed investigation of lymphatic distribu- Histological Staining tion in the interdental papilla connecting the lingual and buccal tissues. To use lymphatic Enzyme histochemical staining vessels as a drug delivery route based on the knowledge of lymphatic architecture and After immersion fixation of the produced distribution, investigation using normal serial sections in 100% ethanol. sections were human samples is essential since there may rinsed with a tris-maleate buffer solution be morphological differences between humans (pH 7.2) and immersed in 5’-nucleotidase and animals. The purpose of this study is to (5’-Nase) substrate solution at 37ºC for 30 reveal the lymphatic architecture beneath the minutes. After rinsing again with a tris- epithelium in the human interdental papilla maleate buffer solution, sections were using three-dimensional reconstructed images immersed in a 1% ammonium sulfide solution with enzyme histochemical and immuno- for 2 minutes to color the lymphatic vessels histochemical staining. (16). After rinsing with distilled water, sections were mounted in 30% glycerin for MATERIALS AND METHODS observation and image capturing. After confirming distinguishing lymphatic and Sample Preparation blood vessels by alkaline phosphatase staining in some sections, counter staining was Human gingival samples were collected conducted on the remaining sections using from seven patients who visited the perio- hematoxylin. Sections were observed dontal clinic at the Iwate Medical University and photographed after remounting with Hospital. Informed consent was obtained 30% glycerin. from all subjects who participated in this study. This research was initiated upon Immunohistochemical staining approval of the Iwate Medical University School of Dentistry Ethics Committee After immersion fixation of the produced (approval number 01065). Samples used in serial sections in 100% ethanol, serial sections this study consisted of human interdental were rinsed for 5 minutes with PBS solution papilla including the lamina propria obtained (0.01 mol/l, pH 7.4, Mitsubishi Chemical from excision for therapeutic purposes Medience Inc., Tokyo, Japan) at room during periodontal surgery. Since gingival temperature. Repeating this procedure three tissues are usually excised with a margin of times, endogenous peroxidase activity was safety consisting of normal tissue, we defined blocked with 3% H2O2 in PBS and sections Permission granted for single print for individual use. Reproduction not permitted without permission of Journal LYMPHOLOGY. 148 Fig. 1. Histological images of the interdental papilla (hematoxylin and eosin staining). A: Overview image (4x). B: Magnified image of the square in Fig. 1 (10x). Inflammatory cell infiltration can be seen near the upper layer of the lamina propria beneath the epithelium (thick arrow). Connective tissue papillae are shown extending towards the epithelium (thin arrow). EP: epithelium, LP: lamina propria. Fig. 1A ) Insert: Schema of the gingival tissue. Cr: Cervical line, R: Root, Cr: Crown, IDP: Interdental Papilla, FG: Free gingiva, AG: Attached gingiva, FGG: Free gingival groove, Al: Alveolus, AB: Alveolar bone, PL: Periodontal ligament. were rinsed again with PBS. In a moist Japan) with a color, chilled 3CCD camera chamber, Special Block A (ACUITY Biotin (C5810®, Hamamatsu Photonics, Tokyo, Free Mouse-on-Mouse Polymer Detection Japan). The obtained two-dimensional System, Covance, California, USA) was images were directly input to a computer. applied onto sections and a 30 minute reaction Coordinating the axes of the two-dimensional time was allowed at room temperature. images, 5’-Nase positive and D2-40 positive Sections were immersed in a 1:40 dilution of lymphatic vessels were extracted and image D2-40 mouse monoclonal antibody (Covance) processing (dichotomizing) was manually with 1% BSA in PBS in a moist chamber for conducted using Photoshop® CS4 (Adobe, 1 hour at room temperature. After rinsing San Jose, USA). Three-dimensional images of with PBS solution for 5 minutes three times, lymphatic vessels were then reconstructed Special Block B was applied in a moist using 3D visualization software (ZedView®DB, chamber. After 10 minutes of reaction at Ver.6.0, LEXI, Tokyo, Japan) (14,17). room temperature, sections were rinsed for Rotating animations were constructed to 5 minutes three times. After rinsing, the same observe distribution of the lymphatic vessels operation using MoM Polymer Link. Color beneath the epithelium from all directions. was then developed with a DAB substrate kit (Vector Laboratories, Burlingame, USA) RESULTS for 5 minutes at room temperature. Following rinsing with distilled water, sections were Histological Structure counterstained with hematoxylin, and embedded in 30% glycerin. The lamina propria beneath the epithelium of the human interdental papilla Reconstruction of three-dimensional images samples showed an inflammatory cell infiltration in the upper layer. This infil- Serial sections with lymphatic staining tration was largely absent from the deeper were observed and photographed using an layers of the lamina propria (Fig. 1A,B). optical microscope (E1000M®, Nikon, Tokyo, No prominent vasodilatations were observed Permission granted for single print for individual use. Reproduction not permitted

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