Visualization of junctional epithelial cell replacement by oral gingival epithelial cells over a life time and after gingivectomy.

Junctional epithelium (JE), which is derived from odontogenic epithelial cells immediately after eruption, is believed to be gradually replaced by oral gingival epithelium (OGE) over a lifetime. However, the detailed process of replacement remains unclear. The aim of the present study was to clarify the process of JE replacement by OGE cells using a green fluorescent protein (GFP)-positive tooth germ transplantation method. GFP-positive JE was partly replaced by OGE cells and completely replaced on day 200 after transplantation, whereas there was no difference in the expression of integrin ß4 (Itgb4) and laminin 5 (Lama5) between JE before and after replacement by OGE cells. Next, GFP-positive JE was partially resected. On day 14 after resection, the regenerated JE consisted of GFP-negative cells and also expressed both Itgb4 and Lama5. In addition, the gene expression profile of JE derived from odontogenic epithelium before gingivectomy was partly different from that of JE derived from OGE after gingivectomy. These results suggest that JE derived from the odontogenic epithelium is gradually replaced by OGE cells over time and JE derived from the odontogenic epithelium might have specific characteristics different to those of JE derived from OGE.

Expression of cytokeratins in enamel organ, junctional epithelium and epithelial cell rests of Malassez.

BACKGROUND AND OBJECTIVE: After tooth formation is complete, it is suggested that continuity exists between the epithelial cell rests of Malassez (ERM), reduced enamel epithelium (REE) and subsequently the junctional epithelium. However, the junctional epithelium was reported to differ from REE and ERM. The developmental relationships between and among them remain controversial. Therefore, in the present study we examined the expression of cytokeratins in the three types of epithelia to investigate the epithelial phenotypes. MATERIAL AND METHODS: The maxillae of Wistar rats, 1, 2, 3 and 7 wk of age, were used, and the expression of CK14, CK17, CK19, CK10/CK13 and AE1/AE3 was detected using immunoperoxidase techniques. RESULTS: There was negative staining for CK10/CK13 in all the epithelia. ERM stained strongly for AE1/AE3, CK14, CK17 and CK19. During the transformation of inner enamel epithelial (IEE) cells into reduced ameloblasts and subsequently into junctional epithelium, strong staining for CK14 was evident in IEE, REE and junctional epithelium, whereas the expression of AE1/AE3 and of CK19 were initially negative in IEE and then strong in REE and junctional epithelium, respectively. In particular, the expression of CK17 was strongly positive in ERM and REE, but was negative in IEE and junctional epithelium. CONCLUSION: ERM are of odontogenic origin and junctional epithelium has an epithelial phenotype different from REE and ERM. This is the first report to demonstrate that CK17 can be used as a marker to distinguish junctional epithelium from ERM.

Morphological and functional characteristics of human gingival junctional epithelium.

BACKGROUND: This study aims to observe the morphological characteristics and identify the function characteristics of junctional epithelium (JE) tissues and cultured JE cells. METHODS: Paraffin sections of human molar or premolar on the gingival buccolingual side were prepared from 6 subjects. HE staining and image analysis were performed to measure and compare the morphological difference among JE, oral gingival epithelium (OGE) and sulcular epithelium (SE). Immunohistochemistry was applied to detect the expression pattern of cytokeratin 5/6, 7, 8/18, 10/13, 16, 17, 19, and 20 in JE, OGE and SE. On the other hand, primary human JE and OGE cells were cultured in vitro. Cell identify was confirmed by histology and immunohistochemistry. In a co-culture model, TEM was used to observe the attachment formation between JE cells and tooth surface. RESULTS: Human JE was a unique tissue which was different from SE and OGE in morphology. Similarly, morphology of JE cells was also particular compared with OGE cells cultured in vitro. In addition, JE cells had a longer incubation period than OGE cells. Different expression of several CKs illustrated JE was in a characteristic of low differentiation and high regeneration. After being co-cultured for 14 d, multiple cell layers, basement membrane-like and hemidesmosome-like structures were appeared at the junction of JE cell membrane and tooth surface. CONCLUSIONS: JE is a specially stratified epithelium with low differentiation and high regeneration ability in gingival tissue both in vivo and in vitro. In co-culture model, human JE cells can form basement membrane-like and hemidesmosome-like structures in about 2 weeks.

Allogeneic stem cells from deciduous teeth in treatment for periodontitis in miniature swine.

BACKGROUND: Regeneration of lost periodontium in periodontitis is a challenge in that alveolar bone, cementum, and periodontal ligament need to be restored to their original architecture. Stem cells from exfoliated deciduous teeth (SHEDs) appear to be an attractive candidate for periodontium tissue regeneration. Previously, the authors successfully regenerated periodontal defects using autologous and allogeneic periodontal ligament stem cells (PDLSCs). The purpose of the present study is to investigate the ability of allogeneic SHEDs to regenerate lost periodontium in a swine periodontitis model. METHODS: Animal models of periodontitis were established in miniature pigs, and allogeneic stem cells were isolated from miniature pig deciduous teeth (SPDs). The animal models were treated with SPDs plus hydroxyapatite/tricalcium phosphate (HA/TCP). Allogeneic PDLSCs plus HA/TCP or HA/TCP alone were set as positive control or control, respectively. Clinical assessments, computed tomography (CT) scanning, and histologic examination were used to evaluate the outcome of tissue regeneration. RESULTS: Clinical indices including probing depth, gingival recession, and attachment loss showed significant restoration in the SPD and PDLSC treatment groups, compared to the HA/TCP group 12 weeks post-transplantation. Meanwhile, CT scans showed that 75% of the samples had successful hard-tissue regeneration in both PDLSC and SPD groups, compared to the HA/TCP group, where the success rate was only 25%. In addition, histologic examination demonstrated that SPD and PDLSC treatment brought about remarkable regeneration of periodontal tissues, whereas periodontal regeneration was rare in the HA/TCP group. CONCLUSIONS: Allogeneic SPDs can effectively repair hard and soft tissue loss brought about by periodontitis in a swine model. Allogeneic SHEDs, which are easily accessible, may be applied to treat periodontitis in clinics in the future.

Morphogenesis of peri-implant mucosa revisited: an experimental study in humans.

AIM: To apply a novel human model to evaluate the morphogenesis of the mucosal attachment to implants. MATERIAL AND METHODS: Twenty one patients receiving implant-supported single-tooth replacement were enrolled in this study. After implant installation, a custom-designed experimental abutment was connected to the implant. Soft tissue biopsies representing 2, 4, 8 or 12 weeks of healing were collected by the use of a circular cutting device and prepared for histological analysis. RESULTS: The soft tissue biopsies were retrieved, preserved and processed with a technique that was safe and reproducible. The results from the histological analysis in regards to dimensional and qualitative changes in the mucosa over time were consistent with those reported from animal experiments. At 8 weeks, the soft tissue dimension was about 3.6 mm and included a barrier epithelium of 1.9 mm and a connective tissue portion of 1.7 mm. Similar dimensions were found at 12 weeks. CONCLUSION: It is suggested that the new human model provides advantages in terms of cost-effectiveness in research as well as from ethical aspects and should be considered as an alternative to pre-clinical in vivo studies in animals.

Expression of odontogenic ameloblast-associated and amelotin proteins in the junctional epithelium.

Two novel proteins - odontogenic ameloblast-associated protein and amelotin - have recently been identified in maturation-stage ameloblasts and in the junctional epithelium. This article reviews the structure and function of the junctional epithelium, the pattern of expression of odontogenic ameloblast-associated and amelotin proteins and the potential involvement of these proteins in the formation and regeneration of the junctional epithelium.

Biomimetic surface modification using synthetic oligopeptides for enhanced guided bone regeneration in beagles.

BACKGROUND: In previous studies, oligopeptide corresponding to the cell-binding domains of bone morphogenetic proteins that bind to bone morphogenetic protein receptor enhanced the bone regenerative capacity of bovine bone minerals (BBM). The aim of this study is to evaluate the ability of BBM coated with oligopeptide to promote periodontal regeneration in a 1-wall intrabony defect model in dogs. METHODS: The second and third mandibular premolars and first molars of six adult beagles were extracted bilaterally, and the extraction sites were allowed to heal for 10 weeks. The 1-wall intrabony defects were prepared bilaterally on the mesial and distal side of the fourth mandibular premolars. Twenty-four intrabony defects were assigned to four treatment groups: 1) open flap debridement; 2) guided tissue regeneration (GTR); 3) GTR with a collagen membrane and BBM; and 4) GTR with a collagen membrane and BBM coated with the oligopeptide (Pep-BBM). The animals were sacrificed 10 weeks after surgery. For the histometric analysis, defect height, junctional epithelium migration, new cementum, new bone height, and new bone area were measured. New bone volume was measured using microcomputed tomography. RESULTS: Wound healing was generally uneventful. For junctional epithelium migration, the BBM and Pep-BBM groups exhibited mean (± SE) values of 0.53 ± 0.41 and 0.48 ± 0.30 mm, and for new cementum height, 1.71 ± 0.46 and 2.50 ± 2.00 mm, respectively. For junctional epithelium migration and cementum regeneration, there were no significant differences between the two groups. The mean (± SE) values of new bone height and new bone volume in the Pep-BBM group (3.88 ± 0.31 mm and 32.35% ± 9.60%) were significantly greater than the mean values for the BBM group (2.60 ± 0.41 mm and 20.56% ± 1.89%). For bone regeneration, the Pep-BBM group showed superior results compared to the BBM group with statistically significant differences. CONCLUSIONS: Through various parameters to evaluate periodontal regeneration, this oligopeptide coating influenced only the ability of BBM to promote bone regeneration in 1-wall intrabony defects in beagles. Junctional epithelium migration and cementum regeneration were not affected by this oligopeptide coating, and further investigations with special focus on regeneration of the periodontal ligament are necessary.

Epithelial integrins with special reference to oral epithelia.

Adhesion of epithelium to the extracellular matrix is crucial for the maintenance of systemic and oral health. In the oral cavity, teeth or artificial dental implants penetrate the soft tissue of the gingiva. In this interface, gingival soft tissue needs to be well attached via the epithelial seal to the tooth or implant surface to maintain health. After injury or wounding, epithelial tissue rapidly migrates to form the initial epithelial cover to restore the barrier against infection. These events are crucially dependent on deposition of extracellular matrix and proper activation and function of integrin receptors in the epithelial cells. Recent experimental evidence suggests that epithelial integrins also participate in the regulation of periodontal inflammation. In this review, we will discuss the structure and function of epithelial integrins and their extracellular ligands and elaborate on their potential role in disease and repair processes in the oral cavity.

In vivo investigation on connective tissue healing to polished surfaces with different surface wettability.

OBJECTIVES: Connective tissue in contact to transgingival/-dermal implants presents itself as tight scar formation. Although rough surfaces support the attachment they increase bacterial colonisation as well. In contrast to surface roughness, little is known about the influence of surface wettability on soft-tissue healing in vivo. We therefore investigated the influence of different surface wettabilities on connective tissue healing at polished implant surfaces in vivo. MATERIAL AND METHODS: Three polished experimental groups (titanium, titanium coated with hydrophobic nano-crystalline diamond (H-NCD) and titanium coated with hydrophilic nano-crystalline diamond (O-NCD) were inserted into the subcutaneous connective tissue of the abdominal wall of 24 rats. Animals were sacrificed after 1 and 4 weeks resulting in eight specimen per group per time point. Specimen were subjected to histological evaluation (van Giesson's staining) and immunohistochemistry staining for proliferating cell nuclear antigen (PCNA), fibronectin and tumour necrosis factor-alpha (TNF-α). RESULTS: Histological evaluation revealed dense scar formation at the titanium and H-NCD surfaces. In contrast, the connective tissue was loose at the O-NCD surface with a significantly higher number of cells after 4 weeks. O-NCD demonstrated a strong expression of PCNA and fibronectin but a weak expression of TNF-α. In contrast, the PCNA and fibronectin expression was low at the titanium and H-NCD, with a strong signal of TNF-α at the H-NCD surface. CONCLUSIONS: Hydrophilicity influences the connective tissue healing at polished implant surfaces in vivo positively. The attachment of connective tissue and the number of cells in contact to the surface were increased. Moreover, the inflammatory response is decreased at the hydrophilic surface.

Soft tissue considerations in implant site development.

Healthy soft tissue surrounding a dental implant is essential for health, function, and esthetics. The development of the tooth includes the formation of a biologic connection between the living tissues that has to be created during the healing process after placement of the implant. The success of dental implants is dependent on the establishment of a soft-tissue barrier that is able to shelter the underlying osseous structures and the osseointegration surrounding the implant body. The esthetics of a dental implant prosthesis depends on the health and stability of the peri-implant mucosa. Understanding of soft-tissue healing and maintenance around dental implants is paramount for implant success. This article discusses the soft-tissue interface, aspects of soft-tissue health, and esthetics during treatment planning and therapy.

Expression and function of laminin and integrins on adhesion/migration of primary culture cells derived from rat oral epithelium.

BACKGROUND AND OBJECTIVE: It remains controversial whether or not the junctional epithelium cells that are directly attached to teeth migrate on the enamel surface, as those cells are able to adhere firmly to the enamel. The aim of this study was to investigate the expression patterns of laminin gamma(2), integrin beta(4) and integrin alpha(3), and to examine their potential function in cell migration. MATERIAL AND METHODS: Oral epithelium cells obtained from Sprague-Dawley rats were established in primary culture. We employed a wound-healing assay to characterize the direction of cell extension at the start of cell migration, and observed different localizations of laminin and integrins using immunofluorescence. For functional analyses of integrins, we employed a phosphatidylinositol-3-kinase (PI3K) activator to promote integrin beta(4) function and used P1B5 to inhibit integrin alpha(3) function, and we analyzed the percentage of re-epithelialization as the migration function. RESULTS: Marked accumulation of laminin gamma(2) was detected in the peripheral cytoplasm of cells adjacent to the wound area, as shown by the results of the migration assay. Integrin beta(4) was detected in the distal cell processes of actively migrating cells, while integrin alpha(3) was found in cell membranes of cells adjacent to the wound area. In the functional analyses, the percentage of re-epithelialization was significantly lower in the PI3K-activator group and in the P1B5-treated group (2.5% and 7.2%, respectively) than in the control group (39.0%) (p < 0.01). CONCLUSION: The results suggest that laminin gamma(2) is secreted as a foothold for cell migration, that integrin beta(4) participates in cell adhesion and that integrin alpha(3) is involved in cell migration in the primary culture cells.

Morphogenesis of the peri-implant mucosa: a comparison between flap and flapless procedures in the canine mandible.

OBJECTIVE: Although it has been shown that the exclusion of the mucoperiosteal flap can prevent postoperative bone resorption associated with flap elevation, there have been only a few studies on the peri-implant mucosa following flapless implant surgery. The purpose of this study was to compare the morphogenesis of the peri-implant mucosa between flap and flapless implant surgeries by using a canine mandible model. STUDY DESIGN: In six mongrel dogs, bilateral edentulated flat alveolar ridges were created in the mandible. After 3 months of healing, 2 implants were placed in each side by either the flap or the flapless procedure. Three months after implant insertion, the peri-implant mucosa was evaluated by using clinical, radiologic, and histometric parameters, which included the gingival index, bleeding on probing, probing pocket depth, marginal bone loss, and the vertical dimension of the peri-implant tissues. RESULTS: The height of the mucosa, length of the junctional epithelium, gingival index, bleeding on probing, probing depth, and marginal bone loss were all significantly greater in the dogs that had the flap procedure than in those that had the flapless procedure (P < .05). CONCLUSION: These results indicate that gingival inflammation, the height of junctional epithelium, and bone loss around nonsubmerged implants can be reduced when implants are placed without flap elevation.

Establishment and characterization of immortalized human gingival keratinocyte cell lines.

BACKGROUND AND OBJECTIVE: Primary human keratinocytes are used to analyze the properties of the oral epithelium and the early stages of oral bacterial infections. In vitro, these cells are characterized by their short life span and restricted availability. Approaches for culturing these cells will end after approximately 6-10 passages as a result of entry into apoptosis. For this reason, it is important to generate cell lines suitable for obtaining an unlimited source of cells. Therefore, the aim of the present study was to generate gingival keratinocyte cell lines and to compare their in vitro behaviour with those of primary human gingival keratinocytes. MATERIAL AND METHODS: Primary human gingival keratinocytes were immortalized with a combination of the human papilloma virus onkoproteins E6 and E7. The pattern of the cytokeratins, involucrin and filaggrin was investigated by intracellular staining using flow cytometry. This method allows quantitative analysis of the expression of a variety of intracellular or extracellular markers. RESULTS: The immortalized cell lines showed many morphological similarities, expressing a cytokeratin pattern that is comparable with that of primary gingival keratinocytes. Furthermore, they developed transepithelial electrical resistance, which is a marker for the generation of tight junctions. These results indicate that the cells might be able to act as an epithelial barrier, reflecting the reaction of primary human cells. CONCLUSION: The establishment of a continuous line of human gingival epithelial cells with functional characteristics of the epithelial barrier provides a valuable in vitro model for using to study the early steps of gingival/periodontal infections.

The effects of laser microtextured collars upon crestal bone levels of dental implants.

PURPOSE: The purpose of this study was to examine the crestal bone, connective tissue, and epithelial cell response to a laser microtextured collar compared with a machined collar, in the dog model. MATERIALS: Six mongrel dogs had mandibular premolars and first molars extracted and after healing replaced with BioLok implants 4 x 8 mm. Each dog had 3 control implants placed on one side of the mandible and 3 experimental, laser microtextured, implants placed contralaterally. After 3 months, 1 dog was killed. Bridges were placed on the implants of 4 of the dogs. The sixth dog served as a negative control for the duration of the experiment. Two of the dogs were killed 3 months after loading, of the dogs were killed 6 months after loading as was the negative (unloaded) control. Histology, electron microscopy, and histomorpho-metric analysis was done on histologic sections obtained from block sections of the mandible containing the implants. RESULTS: Initially the experimental implants showed greater bone attachment along the collar. With time the bone heights along the control and experimental collars were equivalent. However, the controls had more soft tissue downgrowth, greater osteoclastic activity, and increased saucerization compared with sites adjacent to experimental implants. There was closer adaptation of the bone to the laser microtextured collars. CONCLUSION: Use of tissue-engineered collars with microgrooving seems to promote bone and soft tissue attachment along the collar and facilitate development of a biological width.

Preprosthetic periodontal surgery in the proximal area with modification of the col area: results following the reestablishment of the contact point.

BACKGROUND: Anatomic characteristics of interproximal areas are dependent on the anatomy, position, and proximal contact of adjacent teeth. The objective of this study was to investigate the influence of the reestablishment of the interproximal contact following the restorative alveolar interface (RAI) procedure on the interproximal gingival COL and formation of the interdental gingival papilla. METHODS: Six mongrel dogs received bilateral apically positioned flaps, crown lengthening, and the RAI procedure on the maxillary fourth bicuspid and first molar. After 2 weeks, in a randomized manner, one side was prepared to receive metallic crowns and the opposite side remained as the control. The crowns were cemented at the 4-week postoperative period, and the dogs were sacrificed after another 4 weeks, totaling a period of 4 weeks with the full crowns in position and a total of 8 postoperative weeks. Histologic specimens were stained with hematoxylin and eosin and Mallory dyes. Sections 6 micro m thick were obtained in a bucco-lingual plane allowing ample visualization of the interproximal area. RESULTS: Clinical measurements revealed that, in the restored sides, four animals had complete fill of the interdental spaces with gingival papilla, whereas the other two dogs had a distance from the contact point to the tip of papilla varying from 0.02 to 0.021 mm. In the control group, papillae were totally reepithelialized with keratinized epithelium and a convex form. The epithelium completely covered the connective tissue and showed both epithelial projections and surface desquamation. On the test group, despite the presence of the prosthesis, the COL morphology modified by preprosthetic surgery was not altered, presenting a convex papilla with a triangular form and with a keratinized epithelium. Additional histologic characteristics were the same as found in the control group. CONCLUSION: Based on the results of this study, the reestablishment of the contact point does not revert what was obtained with the RAI procedure; the interproximal tissues remain convex and keratinized.

Root coverage in a class IV recession defect achieved by creeping attachment: a case report.

BACKGROUND AND OBJECTIVES: The amount of root coverage obtained after a graft procedure may be improved after the early phase of healing by a coronal displacement of the gingival attachment. The aim of this report is to present a clinical case of complete root coverage of a Miller's class IV recession achieved by creeping attachment subsequent to a laterally repositioned flap. METHODS: In 1995, a 44-year-old male patient was referred for a root coverage graft on the upper right central incisor. Clinical examination revealed that the upper right central incisor had a recession of 7 mm. The defect was classified as class IV according to Miller's classification of marginal tissue recession. It was decided that root coverage would be attempted using a laterally repositioned flap from the upper right lateral incisor and upper right canine. Sutures were removed ten days after surgery. RESULTS: Four months after grafting, the amount of root coverage obtained was 4 mm. After an 8-year period, the previously denuded root surfaces were entirely covered by soft tissue. The marginal position of the gingiva appeared stable, the gingival tissue became firmly attached to the root surface and probing showed a shallow sulcular depth. CONCLUSION: Several interesting observations were made after 8 years. In conclusion, the most significant and interesting finding of this report is that the amount of interdental papilla and marginal gingival tissue covering donor and recipient areas improved with time, providing an excellent aesthetic appearance.

Tissue healing in implants immediately placed into postextraction sockets: a pilot study in a mini-pig model.

OBJECTIVE: A pilot in vivo study was conducted to evaluate (1) the rate of osseointegration at apical, middle, and coronal levels of oral implants immediately installed into fresh extraction sockets; (2) the maturation of the newly formed bone surrounding implants during 60 days of healing; and (3) the epithelium seal development. STUDY DESIGN: The premolars of 8 male adult mini-pigs were extracted at each mandibular site under general anesthesia. In the experimental side, Frialit-2 implants were immediately inserted. The gap between bone and implants ranged between 3 and 6 mm circumferencially. Bone specimens were obtained at 7, 15, 30, and 60 days after surgery for histologic and histomorphometric studies. Bone-to-implant contact (BIC), bone volume, trabecular thickness, number, and separation were recorded. Nonparametric exact tests were used to evaluate data. RESULTS: BIC at the coronal level was observed close to 0% at day 7 and increased up to 60% at day 60 after surgery on an average. BIC increased from 11.7% to 47.38% at middle level and from 53.4% to 67.38% at apical level from day 7 to day 60. With respect to bone maturation, in the earlier stages of healing, many thin trabeculae were observed, which, particularly at coronal level, became significantly fewer and thicker in more advanced stages. At day 60, the features of the bone were similar to those of baseline. The epithelium never migrated more than 1.8 mm apically to the top of the alveolar bone level. CONCLUSION: When implants are placed immediately into fresh extraction sockets, in minipig models osseointegration also occurs without initial bone contact.

Bioactive Glass Efficacy in the Periodontal Healing of Intrabony Defects in Monkeys

A proposta desse estudo foi avaliar histologicamente a eficácia de um vidro bioativo com pequena variação de tamanho de partículas (Biogran) na cicatrização periodontal de defeitos infra-ósseos de 2 paredes em macacos. Os defeitos foram feitos na mesial dos segundos pré-molares direito e esquerdo de 4 macacos, a seguir foram preenchidos com guta-percha e, após 15 dias, foram debridados e preenchidos naturalmente com coágulo (controle) ou com vidro bioativo (teste). Nos sítios-controle, o epitélio juncional migrou até a base do defeito. A formação de novo cemento foi mais significante nos defeitos-teste. Ambos os tipos de defeitos, controle e teste, apresentaram formação de novo osso na área da base dos defeitos. Os defeitos-teste apresentaram deposição de novo osso ao redor e dentro de partículas de vidro bioativo localizadas no terço médio, distantes das paredes do defeito. A análise histológica demonstrou que o vidro bioativo com partículas de 300 a 355 µm favoreceu nova inserção periodontal. Concluiu-se que o vidro bioativo testado teve melhor potencial de cura que o debridamento. O material enxertado mostrou promissora inibição da migração apical do epitélio junctional e maior deposição de cemento na superfície radicular em defeitos infra-ósseos A substituição das partículas de vidro bioativo por novo osso ocorreu devido não apenas a uma atividade osteo-condutora, mas também a uma capacidade osteo-estimuladora. Futuras investigações devem avaliar esse potencial comparativamente a outros materiais de enxerto, técnicas regenerativas e modificadores biológicos, assim como, avaliar longitudinalmente a estabilidade dessa nova inserção.