OBJECTIVE: To evaluate the type of wound healing following modified crown lengthening surgery in dog model to provide a biological basis for its clinical application. METHODS: Flap surgery, traditional crown lengthening procedure and modified crown lengthening procedure were performed on the right maxillary central incisor, the left maxillary central incisor and the left maxillary first lateral incisor respectively of five male beagle dogs. The right maxillary first lateral incisors with no surgical intervention were used as controls. Thirty-six weeks after the experimental procedure, tissue blocks were harvested and prepared for histological examination and analysis. RESULTS: Histometric examination of buccolingual sections stained with hematoxylin-eosin demonstrated that the type of wound healing in the flap surgery group was re-attachment, similar to the control group. For the traditional crown lengthening surgery group, all of the five beagle dogs had lamellar cementum defects on root surface, the wound healing of four beagle dogs was new attachment accompanied by new cementum formation at cementum defect areas and the suprac-restal connective tissue was functionally oriented perpendicular to the new cementum. The wound healing of the other beagle dog was long junctional epithelial attachment, in which the junctional epithelium extended to the apical terminus of the cementum defect. In the modified crown lengthening surgery group, four beagle dogs had cementum defects on root surface (two lamellar cementum defects and two shallow platform-like cementum defects), the wound healing of three beagle dogs was new attachment, however, the supracrestal connective tissue was parallel to the root surface. The type of wound healing of another one beagle dog was long junctional epithelial attachment. Wound healing of one beagle dog in this group could not be characterized due to incomplete dissection. CONCLUSION Wound healing in the modified crown lengthening surgery group was similar to the traditional crown lengthening surgery group, and two types of wound healing were observed: new attachment and long junctional epithelium attachment. Neither type of root treatment procedure (root planing or root reshaping) nor root surface defect pattern (the lamellar cementum defect or shallow platform-like cementum defect) influenced the observed type of wound healing.
Animals ; Connective Tissue ; Crown Lengthening ; Dogs ; Eosine Yellowish-(YS) ; Epithelial Attachment/pathology* ; Hematoxylin ; Male ; Tooth Root/surgery* ; Wound Healing

OBJECTIVE: Calprotectin, the heterdimer of S100A8 and S100A9, is the major cytoplasmic protein of neutrophils, which is also expressed or induced in gingival epithelial cells, activated mononuclear macrophages and vascular endothelial cells. Calprotectin is intimately associated with the initiation and progression of periodontitis, but the in vivo expression patterns of calprotectin in healthy and inflamed periodontal tissue are not fully understood. To observe the expression, distribution and cellular localization of calprotectin in the samples of healthy periodontal tissues and experimental periodontitis tissues of Beagles and to explore their relationship with periodontal inflammation and possible effect. METHODS: Experimental periodontitis model was established by ligation around the mandibular second molar of the Beagle dogs, while the contralateral teeth were healthy controls. Induction duration was 12 weeks, before the dogs were executed. Tissue specimens were demineralized and serial sections were made conventionally. The in vivo expression of calprotectin in the healthy and inflamed periodontal tissues were examined by immunohistochemistry. The in vitro expression of calprotectin in human primary gingival fibroblasts (GFs) and periodontal ligament (PDL) cells were detected by immunocytochemistry. RESULTS: Immunohistochemistry analysis indicated that calprotectin was expressed in gingival epithelial cells and infiltrated neutrophils in the healthy periodontium within the gingival epithelium, S100A8/A9 was most strongly expressed in the junctional epithelium, followed by surface epithelium, and least expressed in the sulcular epithelium. The S100A8/A9 expression levels were sharply defined at the junction between the junctional epithelium and the sulcular epithelium. In periodontal inflammatory lesions, the expression level of calprotectin in sulcular epithelium and junctional epithelium was up-regulated than that in the healthy gingival epithelium. Calprotectin was inducibly expressed in fibroblast-like cells in gingival connective tissue and periodontal ligament tissue, microvascular endothelial cells (ECs) and bone marrow fibroblasts under inflammatory conditions. Additionally, the expression of calprotectin in primary human GFs and PDL cells was confirmed by immunnocytochemistry staining. CONCLUSION Constitutively expressed in neutrophils and gingival epithelial cells, and calprotectin might maintain the homeostasis and integrity of periodontium. Inflammation-induced expression of calprotectin in GFs, PDL cells, microvascular ECs and bone marrow fibroblasts might process anti-microbial function and promote leukocytes transmigration to defend the host against the microorganisms.
Animals ; Dogs ; Endothelial Cells ; Epithelial Attachment ; Gingiva ; Humans ; Leukocyte L1 Antigen Complex ; Periodontium

PURPOSE: Despite the well-known anti-inflammatory effects of vitamin D in periodontal health, its mechanism has not been fully elucidated. In the present study, the effect of vitamin D on strengthening E-cadherin junctions (ECJs) was explored in human gingival keratinocytes (HGKs). ECJs are the major type of intercellular junction within the junctional epithelium, where loose intercellular junctions develop and microbial invasion primarily occurs. METHODS: HOK-16B cells, an immortalized normal human gingival cell line, were used for the study. To mimic the inflammatory environment, cells were treated with tumor necrosis factor-alpha (TNF-α). Matrix metalloproteinases (MMPs) in the culture medium were assessed by an MMP antibody microarray and gelatin zymography. The expression of various molecules was investigated using western blotting. The extent of ECJ development was evaluated by comparing the average relative extent of the ECJs around the periphery of each cell after immunocytochemical E-cadherin staining. Vitamin D receptor (VDR) expression was examined via immunohistochemical analysis. RESULTS: TNF-α downregulated the development of the ECJs of the HGKs. Dissociation of the ECJs by TNF-α was accompanied by the upregulation of MMP-9 production and suppressed by a specific MMP-9 inhibitor, Bay 11-7082. Exogenous MMP-9 decreased the development of ECJs. Vitamin D reduced the production of MMP-9 and attenuated the breakdown of ECJs in the HGKs treated with TNF-α. In addition, vitamin D downregulated TNF-α-induced nuclear factor kappa B (NF-κB) signaling in the HGKs. VDR was expressed in the gingival epithelium, including the junctional epithelium. CONCLUSIONS: These results suggest that vitamin D may avert TNF-α-induced downregulation of the development of ECJs in HGKs by decreasing the production of MMP-9, which was upregulated by TNF-α. Vitamin D may reinforce ECJs by downregulating NF-κB signaling, which is upregulated by TNF-α. Strengthening the epithelial barrier may be a way for vitamin D to protect the periodontium from bacterial invasion.
Bays ; Blotting, Western ; Cadherins ; Cell Line ; Down-Regulation ; Epithelial Attachment ; Epithelium ; Gelatin ; Humans ; Intercellular Junctions ; Keratinocytes ; Matrix Metalloproteinase 9 ; Matrix Metalloproteinases ; NF-kappa B ; Periodontium ; Receptors, Calcitriol ; Tumor Necrosis Factor-alpha ; Up-Regulation ; Vitamin D ; Vitamins

Epithelial attachment via the basal lamina on the tooth surface provides an important structural defence mechanism against bacterial invasion in combating periodontal disease. However, when considering dental implants, strong epithelial attachment does not exist throughout the titanium-soft tissue interface, making soft tissues more susceptible to peri-implant disease. This study introduced a novel synthetic peptide (A10) to enhance epithelial attachment. A10 was identified from a bacterial peptide display library and synthesized. A10 and protease-activated receptor 4-activating peptide (PAR4-AP, positive control) were immobilized on commercially pure titanium. The peptide-treated titanium showed high epithelial cell migration ability during incubation in platelet-rich plasma. We confirmed the development of dense and expanded BL (stained by Ln5) with pericellular junctions (stained by ZO1) on the peptide-treated titanium surface. In an adhesion assay of epithelial cells on A10-treated titanium, PAR4-AP-treated titanium, bovine root and non-treated titanium, A10-treated titanium and PAR4-AP-treated titanium showed significantly stronger adhesion than non-treated titanium. PAR4-AP-treated titanium showed significantly higher inflammatory cytokine release than non-treated titanium. There was no significant difference in inflammatory cytokine release between A10-treated and non-treated titanium. These results indicated that A10 could induce the adhesion and migration of epithelial cells with low inflammatory cytokine release. This novel peptide has a potentially useful application that could improve clinical outcomes with titanium implants and abutments by reducing or preventing peri-implant disease.
Amino Acid Sequence ; Animals ; Benzeneacetamides ; chemical synthesis ; pharmacology ; Cattle ; Cell Adhesion ; drug effects ; Cell Movement ; drug effects ; Cells, Cultured ; Cytokines ; metabolism ; Dental Implants ; Enzyme-Linked Immunosorbent Assay ; Epithelial Attachment ; drug effects ; Epithelial Cells ; cytology ; metabolism ; Microscopy, Confocal ; Microscopy, Electron, Scanning ; Piperidones ; chemical synthesis ; pharmacology ; Platelet-Rich Plasma ; Receptors, Thrombin ; Surface Properties ; Titanium ; chemistry

PURPOSE: The entry of bacteria or harmful substances through the epithelial seal of human gingival keratinocytes (HGKs) in the junctional epithelium (JE) is blocked by specialized intercellular junctions such as E-cadherin junctions (ECJs). However, the influence of roughened substrates, which may occur due to apical migration of the JE, root planing, or peri-implantitis, on the development of the ECJs of HGKs remains largely unknown. METHODS: HGKs were cultured on substrates with varying levels of roughness, which were prepared by rubbing hydrophobic polystyrene dishes with silicon carbide papers. The activity of c-Jun N-terminal kinase (JNK) was inhibited with SP600125 or by transfection with JNK short hairpin RNA. The development of intercellular junctions was analyzed using scanning electron microscopy or confocal laser scanning microscopy after immunohistochemical staining of the cells for E-cadherin. The expression level of phospho-JNK was assessed by immunoblotting. RESULTS: HGKs developed tight intercellular junctions devoid of wide intercellular gaps on smooth substrates and on rough substrates with low-nanometer dimensions (average roughness [Ra]=121.3±13.4 nm), although the ECJs of HGKs on rough substrates with low-nanometer dimensions developed later than those of HGKs on smooth substrates. In contrast, HGKs developed short intercellular junctions with wide intercellular gaps on rough substrates with mid- or high-nanometer dimensions (Ra=505.3±115.3 nm, 867.0±168.6 nm). Notably, the stability of the ECJs was low on the rough substrates, as demonstrated by the rapid destruction of the cell junction following calcium depletion. Inhibition of JNK activity promoted ECJ development in HGKs. JNK was closely associated with cortical actin in the regulation of ECJs in HGKs. CONCLUSIONS: These results indicate that on rough substrates with nanometer dimensions, the ECJs of HGKs develop slowly or defectively, and that this effect can be reversed by inhibiting JNK.
Actins ; Bacteria ; Cadherins* ; Calcium ; Dental Implants ; Epithelial Attachment ; Humans* ; Immunoblotting ; Intercellular Junctions ; JNK Mitogen-Activated Protein Kinases ; Keratinocytes* ; Microscopy, Confocal ; Microscopy, Electron, Scanning ; Peri-Implantitis ; Periodontal Diseases ; Polystyrenes ; Re-Epithelialization ; RNA, Small Interfering ; Root Planing ; Silicon ; Transfection

Inflammation from chronic and acute infections of distal organs and tissues such as periodontitis is a risk factor for atherosclerotic vascular processes. Recently, a new model of atherosclerosis with vascular pathologies was developed in the Mongolian gerbil. In this study, we attempted to develop a model of ligature-induced periodontitis in gerbils and compared the characteristics of that periodontitis model with that in rats and mice. Each gerbil, rat, and mouse was randomly assigned to groups of control and periodontitis. A thread was placed around the cervix of the right and left first molars in the mandible with knots placed on the mesial side of each molar. At day 14 after the ligation, the animals were sacrificed and their mandibles were dissected. To measure alveolar bone loss along with inflammation, histopathological and micro-CT analyses were carried out. Gerbils showed tooth characteristics of deeper gingival crevice, longer cusp, longer root trunk and shorter root than those of rats and mice. The increased CEJ-ABC distance in distal and PDL area in furcation was also observed in ligated gerbils. An inflammatory response in the connective tissue under the junctional epithelium was also shown in all the animals. As a result, we confirmed the induction of periodontitis by ligature in the gerbils. We therefore consider the gerbil to be a useful model for investigating relationship between periodontitis and vascular disease in the same animal.
Alveolar Bone Loss ; Animals* ; Atherosclerosis ; Cervix Uteri ; Connective Tissue ; Epithelial Attachment ; Female ; Gerbillinae* ; Inflammation ; Ligation ; Mandible ; Mice ; Models, Animal* ; Molar ; Pathology ; Periodontitis* ; Rats ; Risk Factors ; Tooth ; Vascular Diseases

PURPOSE: The purpose of this study was to investigate the effects of implant collar design on marginal bone change and soft tissue response by an animal test MATERIALS AND METHODS: Two types of Implant (Neobiotech Co. Seoul, Korea) that only differs in collar design were planted on two healthy Beagle dogs. The implants were divided into two groups, the first group with a beveled collar (Bevel Group) and the second group with "S" shaped collar (Bioseal group). Standardized intraoral radiographs were used to investigate the mesio-distal change of the marginal bone. Histological analysis was done to evaluate the bucco-lingual marginal bone resorption and the soft tissue response adjacent to the implant. Mann-Whitney test was done to compare the mesio-distal marginal bone change at equivalent time for taking the radiographs and the tissue measurements between the groups. RESULTS: Radiographic and histological analysis showed that there was no difference in marginal bone change between the two groups (P>.05). Histological analysis showed Bioseal group had more rigid connective tissue attachment than the Bevel group. There was no difference in biological width (P>.05). Bevel group showed significantly longer junctional epithelium attachment and Bioseal group showed longer connective tissue attachment (P<.05). CONCLUSION: For three months there were no differences in marginal bone change between the Bevel group and the Bioseal group. As for the soft tissue adjacent to the implant, Bioseal group showed longer connective tissue attachment while showing shorter junctional epithelium attachment. There were no differences in biologic width.
Animals ; Bone Resorption ; Connective Tissue ; Dogs ; Epithelial Attachment ; Plants ; Root Canal Filling Materials

PURPOSE: Accurate and exact measurement is an important factor for generating meaningful results in any properly designed study. If all the participating examiners are able to yield similar results, it will be possible to evaluate the objective results of the study more easily and quickly. The purpose of this study was to evaluate the intra- and inter-examiner reproducibility of histometric measurements in the intrabony periodontal defect model. METHODS: One wall intrabony defects were surgically created at the distal aspect of the second and the medial aspect of the fourth mandibular premolars in the right and left jaw quadrants in twenty beagle dogs and the defect sites received the following beta-tri calcium phosphate, growth differentiation factor-0, growth differentiation factor-100 and sham surgery. Histometric analysis was performed after 8 weeks. Histometric parameters were recorded and repeated at three months interval by three examiners. Intra- and inter-examiner reproducibility was assessed. RESULTS: Most parameters of all the groups showed high intra- and inter-examiner reproducibility. Parameters including defect height, bone regeneration height, cementum regeneration height, and formation of junctional epithelium yielded inter-examiner correlation > or = 0.9. The intra-examiner reproducibility showed a high result, over 0.9. CONCLUSIONS: Histometric evaluation of the one-wall intra-alveolar periodontal defect model showed high reproducibility not only for a single given examiner but also among the three examiners.
Alveolar Bone Loss ; Animals ; Bicuspid ; Bone Regeneration ; Calcium ; Calcium Phosphates ; Dental Cementum ; Dogs ; Epithelial Attachment ; Jaw ; Regeneration ; Salicylamides

PURPOSE: The purpose of this study was to preliminarily evaluate the influence of diabetes mellitus (DM) on periodontal tissue without establishment of periodontitis. METHODS: Seven-week-old db/db mice were used for the diabetic experimental group and systematically healthy mice of the same age were used as controls. After 1 week of acclimatization, the animals were sacrificed for hard and soft tissue evaluation. The pattern of bone destruction was evaluated by stereomicroscope evaluation with alizarin red staining and radiographic evaluation by microscopic computerized tomography images. Histological evaluation was performed with hematoxylin and eosin stain for evaluation of soft tissue changes. RESULTS: In both stereomicroscope evaluation and radiograph image analysis, aggressive form of bone destruction was observed in diabetic animals when compared to the systematically healthy controls. In histological evaluation, apical migration of junctional epithelium with slight inflammatory cell infiltration was observed with disarrangement of connective tissue fibers. CONCLUSIONS: Within the limits of this study, diabetic animals presented distortion in periodontal attachment and an aggressive bone loss pattern when compared to the healthy controls, suggesting that DM has an independent effect on periodontal tissue destruction irrespective of the presence or absence of periodontal disease.
Acclimatization ; Animals ; Anthraquinones ; Connective Tissue ; Diabetes Mellitus ; Eosine Yellowish-(YS) ; Epithelial Attachment ; Hematoxylin ; Inflammation ; Mice ; Periodontal Diseases ; Pilot Projects

PURPOSE: To prolong the degradation time of collagen membranes, various cross-linking techniques have been developed. For cross-linking, chemicals such as formaldehyde and glutaraldehyde are added to collagen membranes, but these chemicals could adversely affect surrounding tissues. The aim of this study is to evaluate the ability of porous non-chemical cross-linking porcine-derived collagen nanofibrous membrane to enhance bone and associated tissue regeneration in one-wall intrabony defects in beagle dogs. METHODS: The second and third mandibular premolars and the first molars of 2 adult beagles were extracted bilaterally and the extraction sites were allowed to heal for 10 weeks. One-wall intrabony defects were prepared bilaterally on the mesial and distal side of the fourth mandibular premolars. Among eight defects, four defects were not covered with membrane as controls and the other four defects were covered with membrane as the experimental group. The animals were sacrificed 10 weeks after surgery. RESULTS: Wound healing was generally uneventful. For all parameters evaluating bone regeneration, the experimental group showed significantly superior results compared to the control. In new bone height (NBh), the experimental group exhibited a greater mean value than the control (3.04 +/- 0.23 mm/1.57 +/- 0.59, P = 0.003). Also, in new bone area (NBa) and new bone volume (NBv), the experimental group showed superior results compared to the control (NBa, 34.48 +/- 10.21% vs. 5.09 +/- 5.76%, P = 0.014; and NBv, 28.04 +/- 12.96 vs. 1.55 +/- 0.57, P = 0.041). On the other hand, for parameters evaluating periodontal tissue regeneration, including junctional epithelium migration and new cementum height, there were no statistically significant differences between two groups. CONCLUSIONS: Within the limitations of this study, this collagen membrane enhanced bone regeneration at one-wall intrabony defects. On the other hand, no influence of this membrane on periodontal tissue regeneration could be ascertained in this study.
Absorbable Implants ; Adult ; Animals ; Bicuspid ; Bone Regeneration ; Collagen ; Dental Cementum ; Dogs ; Epithelial Attachment ; Formaldehyde ; Glutaral ; Guided Tissue Regeneration ; Hand ; Humans ; Membranes ; Molar ; Regeneration ; Wound Healing