Structure of junctional epithelium is maintained by cell populations supplied from multiple stem cells.

The junctional epithelium (JE) is an epithelial component that attaches directly to the tooth surface and performs the unique function of protecting against bacterial infections; its destruction causes inflammation of the periodontal tissue and loss of alveolar bone. A recent study that used the single-color lineage tracing method reported that JE is maintained by its stem cells. However, the process by which individual stem cells form the entire JE around a whole tooth remains unclear. Using a 4-color lineage tracing method, we performed a detailed examination of the dynamics of individual stem cells that constitute the entire JE. The multicolor lineage tracing method showed that single-color areas, which were derived from each cell color, replaced all the constituent JE cells 168 d after the administration of tamoxifen. The horizontal section of the first molar showed that the single-color areas in the JE expanded widely. We detected putative stem cells at the external basal layer farthest from the enamel. In this study, JE cells that were supplied from different stem cells were visualized as individual monochromatic regions, and the JE around the first molar was maintained by several JE-specific stem cells. These findings indicated that the JE consisted of several cell populations that were supplied from their multiple stem cells and could help to explore the mechanisms involved in periodontal tissue homeostasis.

Recent Advances of Useful Cell Sources in the Periodontal Regeneration.

BACKGROUND: Periodontitis is an inflammatory disease that can result in destruction of the tooth attachment apparatus. Therefore, periodontal tissue regeneration is currently an important focus of research in the field. Approaches using stem cells and reprogrammed cells, such as induced pluripotent stem cells (iPSCs) or trans-differentiated cells, represent the cutting edge in periodontal regeneration, and have led to many trials for their clinical application. Objectives and Results: In this review, we consider all available stem cell sources, methods to obtain the cells, their capability to differentiate into the desired cells, and the extent of their utilization in periodontal regeneration. In addition, we introduce the new concepts of using iPSCs and transdifferentiated cells for periodontal regeneration. Finally, we discuss the promise of tissue engineering for improving cell therapy outcomes for periodontal regeneration. CONCLUSIONS: Despite their limitations, iPSCs and trans-differentiated cells may be promising cell sources for periodontal tissue regeneration. Further collaborative investigation is required for the effective and safe application of these cells in combination with tissue engineering elements, like scaffolds and biosignals.

Immunolocalization of connective tissue growth factor, transforming growth factor-beta1 and phosphorylated-SMAD2/3 during the postnatal tooth development and formation of junctional epithelium.

Connective tissue growth factor (CTGF) is a downstream mediator of transforming growth factor-beta 1 (TGF-ß1) and TGF-ß1-induced CTGF expression is regulated through SMAD pathway. However, there is no literature showing the expression of TGF-ß1-SMAD2/3-CTGF signaling pathway during postnatal tooth development and the formation of junctional epithelium (JE). Hence, we aimed to analyze the localization of TGF-ß1, CTGF and phosphorylated SMAD2/3 (p-SMAD2/3) in the developing postnatal rat molars. Wistar rats were killed at postnatal (PN) 0.5, 3.5, 7, 14 and 21days and the upper jaws were processed for immunohistochemistry. At PN0.5 and PN3.5, weak staining for TGF-ß1 and CTGF was evident in preameloblasts (PA), while moderate to strong staining was seen in odontoblasts (OD), dental papilla (DPL), secretary ameloblasts (SA), preodontoblasts (PO) and polarized odontoblasts (PoO). There was no staining for p-SMAD2/3 in PA, SA, PO and PoO, although strong staining was localized in DPL. OD was initially moderately positive and then negative for p-SMAD2/3. At PN7, intense staining for TGF-ß1 and CTGF was observed in SA, OD, dental pulp (DP) and predentin respectively. p-SMAD2/3 was strongly expressed in DP and moderately expressed in SA and OD. At PN14 and PN21, both reduced enamel epithelium (REE) and JE showed a strong reaction for TGF-ß1 and CTGF. p-SMAD2/3 was intensely and weakly expressed in REE and JE respectively. These data demonstrate that the expression of CTGF, TGF-ß1 and p-SNAD2/3 is tissue-specific and stage-specific, and indicate a regulatory role for a TGF-ß1-SMAD2/3-CTGF signaling pathway in amelogenesis, dentinogenesis and formation of JE.

Periodontal repair following implantation of beta-tricalcium phosphate with different pore structures in Class III furcation defects in dogs.

The aim of this study was to investigate the effect of the pore characteristics of ß-tricalcium phosphate (ß-TCP) on periodontal healing in class III furcation defects in dogs. Two types of ß-TCP were prepared for grafting; 1) a tunnel pipe structure with an inner diameter of 300 µm, and 2) continuous pore structure with interconnected macropores. The furcations of thirty mandibular premolar teeth were implanted with each type of ß-TCP or were left untreated as control. The dogs were sacrificed 8 weeks post-surgery, and healing was evaluated histologically. Downgrowth of junctional epithelium in the tunnel structure group was significantly less than that in the other two groups (p<0.01). There was significantly more new bone formation and new cementum formation in the tunnel structure group than that in the other two groups (p<0.01). These findings suggested that ß-TCP with a tunnel pipe structure promotes periodontal healing in class III furcation defects.

Application of collagen hydrogel/sponge scaffold facilitates periodontal wound healing in class II furcation defects in beagle dogs.

BACKGROUND AND OBJECTIVE: A three-dimensional scaffold may play an important role in periodontal tissue engineering. We prepared bio-safe collagen hydrogel, which exhibits properties similar to those of native extracellular matrix. The aim of this study was to examine the effect of implantation of collagen hydrogel/sponge scaffold on periodontal wound healing in class II furcation defects in dogs. MATERIAL AND METHODS: The collagen hydrogel/sponge scaffold was prepared by injecting collagen hydrogel, cross-linked to the ascorbate-copper ion system, into a collagen sponge. Class II furcation defects (of 5 mm depth and 3 mm width) were surgically created in beagle dogs. The exposed root surface was planed and demineralized with EDTA. In the experimental group, the defect was filled with collagen hydrogel/sponge scaffold. In the control group, no implantation was performed. Histometric parameters were evaluated 2 and 4 wk after surgery. RESULTS: At 2 wk, the collagen hydrogel/sponge scaffold displayed high biocompatibility and biodegradability with numerous cells infiltrating the scaffold. In the experimental group, reconstruction of alveolar bone and cementum was frequently observed 4 wk after surgery. Periodontal ligament tissue was also re-established between alveolar bone and cementum. Volumes of new bone, new cementum and new periodontal ligament were significantly greater in the experimental group than in the control group. In addition, epithelial down-growth was suppressed by application of collagen hydrogel. CONCLUSION: The collagen hydrogel/sponge scaffold possessed high tissue compatibility and degradability. Implantation of the scaffold facilitated periodontal wound healing in class II furcation defects in beagle dogs.

Expression pattern of odontogenic ameloblast-associated and amelotin during formation and regeneration of the junctional epithelium.

The junctional epithelium (JE) adheres to the tooth surface, and seals off periodontal tissues from the oral environment. This incompletely differentiated epithelium is formed initially by the fusion of the reduced enamel organ with the oral epithelium (OE). Two proteins, odontogenic ameloblast-associated (ODAM) and amelotin (AMTN), have been identified in the JE. The objective of this study was to evaluate their expression pattern during formation and regeneration of the JE. Cytokeratin 14 was used as a differentiation marker for oral epithelial cells, and Ki67 for cell proliferation. Immunohistochemistry was carried out on erupting rat molars, and in regenerating JE following gingivectomy. In the reducing enamel organ and in established JE, ODAM and AMTN were present at the cell-tooth interface while only ODAM and CK14 were found throughout the JE. Both were also conspicuously present in cell clusters situated between the erupting tooth and OE. During JE regeneration, ODAM was detected first at the leading wound edge and then in the regenerating JE. Some cell clusters in the subjacent connective tissue were also positive for ODAM. AMTN appeared later and both AMTN and ODAM accumulated at the interface with the tooth. Cytokeratin 14 gradually appeared in the regenerating JE but the cell clusters showed variable labeling. Cells associated with JE formation and regeneration exhibited higher division activity than adjacent epithelial cells. These findings suggest that ODAM and AMTN have a role at the cell-tooth interface, and that ODAM is likely also implicated in cellular events during formation and regeneration of the JE.

Morphogenesis of the peri-implant mucosa: an experimental study in dogs.

PURPOSE: The objective of the present experiment was to study the morphogenesis of the mucosal attachment to implants made of c.p. titanium. MATERIAL AND METHODS: All mandibular premolars were extracted in 20 Labrador dogs. After a healing period of 3 months, four implants (ITI Dental Implant System) were placed in the right and left sides of the mandible. A non-submerged implant installation technique was used and the mucosal tissues were secured to the conical marginal portion of the implants with interrupted sutures. The sutures were removed after 2 weeks and a plaque control program including daily cleaning of the remaining teeth and the implants was initiated. The animals were sacrificed and biopsies were obtained at various intervals to provide healing periods extending from Day 0 (2 h) to 12 weeks. The mandibles were removed and placed in the fixative. The implant sites were dissected using a diamond saw and processed for histological analysis. RESULTS: Large numbers of neutrophils infiltrated and degraded the coagulum that occupied the compartment between the mucosa and the implant during the initial phase of healing. At 2 weeks after surgery, fibroblasts were the dominating cell population in the connective tissue interface but at 4 weeks the density of fibroblasts had decreased. Furthermore, the first signs of epithelial proliferation were observed in specimens representing 1-2 weeks of healing and a mature barrier epithelium occurred after 6-8 weeks of healing. The collagen fibers of the mucosa were organized after 4-6 weeks of healing. CONCLUSION: It is suggested that the soft-tissue attachment to implants placed using a non-submerged installation procedure is properly established after several weeks following surgery.

Syndecan-1 expression during the formation of junctional epithelium.

BACKGROUND: Syndecans are cell surface heparan sulfate proteoglycans (PG) which can bind to and modulate the action of growth factors and extracellular matrix components (ECM). Syndecan- 1 has been shown to play important roles during early tooth development and wound healing and repair. Among diverse cells and tissues that comprise the periodontium, the junctional epithelium (JE) constitutes a region of significant anatomic and clinical importance, but the nature of inductive signals and molecules involved in its formation is still unclear. Therefore, this work examines if syndecan-1 is associated with formation of JE, and the distribution of other syndecan family members in the epithelium. METHODS: In situ hybridization and immunohistochemical techniques were performed using oral tissues from 4-day-old to 10-week-old mice to investigate the expression of syndecan- 1, -2, -3 and -4 mRNAs and their corresponding proteins. RESULTS: Based on in situ hybridization experiments, all syndecan mRNAs were detected in sulcular epithelium (SE), gingival epithelium (GE), and JE with varying intensity and distribution. Syndecan-1 immunostaining was localized on the cell surface while that of syndecan-2 did not show clear membrane localization. Our experiments in the developing tooth demonstrated that syndecan-1 protein followed characteristic patterns of expression during JE formation and that immunoreactivity for syndecan-1 protein decreased with age when JE cells underwent terminal differentiation. CONCLUSION: Results of syndecan-1 mRNA and protein expression patterns suggested that this proteoglycan might be an important molecule during the formation of JE.

Regeneration of the junctional epithelium and connective tissue after transplantation of detergent-processed allo-teeth.

The authors have developed a new artificial dental implant and evaluated it in a dog model in terms of its potential to produce: I) regeneration of junctional epithelium; II) regeneration and attachment of connective tissue. The implants were constructed from allo-teeth. We removed the cell components from the periodontal ligaments of these teeth with a detergent (1% TritonX-100); the remaining acellular periodontal ligament acted as an extracellular matrix upon which regeneration and attachment could proceed. We placed 10 of these implants in the just-extracted sites of three beagle dogs. We observed regeneration of both junctional epithelium and connective tissue at all implant sites after 3 months. The connective tissue was attached in all cases. Use of the acellular periodontal ligament as an extracellular matrix may facilitate regeneration of host periodontal ligament tissue, thus contributing to recovery of host immunological defense and long-term oral function.

Attachment formation following replantation of cultured cells into periodontal defects--a study in minipigs.

Regeneration processes in the periodontium occur by the interaction of different cell populations. It is known that these cells are also capable of forming new periodontal tissue after culture in vitro. The present study investigated whether replanted cultured cells from the periodontium could contribute to attachment formation. Primary cell cultures from alveolar bone and periodontal ligament were obtained from 11 minipigs. Experimentally induced furaction and interdental defects (n = 168) were treated in groups: (a) flap surgery, replantation of alveolar bone cells, and covering of the defects with Teflon membranes (ABC group); (b) flap surgery, replantation of periodontal ligament cells and membranes (PLC group); (c) flap surgery, bone gelatin (carrier material) and membranes (BG group); (d) flap surgery and membranes (NBG group); (e) flap surgery (FS group); and (f) no treatment (NT group). The defects were clinically and histologically (polyfluorochrome labeling) assessed after 10, 30, and 90 days. In the ABC group, initial calcified tissue formation at the roots was apparent after only 8 days. Marked new formation of cementum and alveolar bone and the development of a new attachment were observed after 90 days. In the BG and the NBG groups, wound healing varied depending on membrane healing and the morphology of the defects, which led to significantly poorer and variable results. Similar results were found in the PLC group, although some defects showed extensive cementum and bone formation. Defects in the FS and the NT groups healed largely by epithelialization. The study shows that replantation of cultured alveolar bone cells leads to formation of new cementum and bone, which, in turn, leads to formation of a new attachment. It is likely that the cells stabilize the tissue formation in the defect or on the root surface in the early phase of wound healing and prevent epithelial downgrowth. Results also show that regeneration in the periodontium is determined by the availability of (precursor) cells capable of forming calcified tissues.

The role of epithelium in the development, structure and function of the tissues of tooth support.

The roles of epithelium in the development, structure and function of the tissues of tooth support are reviewed. Epithelium is involved in initiating odontogenesis which includes the tissues of tooth support and this role is discussed. Particular attention is paid to Hertwig's epithelial root sheath and its participation in the formation of the hyaline layer on the root surface as well as its possible role in initiating the differentiation of cementoblasts. The possible functions of the epithelial cell rests are reviewed and it is concluded that as yet no function can be ascribed to them. Evidence for an increasing role for dental epithelium in tooth eruption is presented and the role of dental epithelium in establishing the dentogingival junction is discussed, with the conclusion drawn that this role temporary.

The effects of scaling procedures on epithelial cell growth on titanium surfaces.

This study examined in vitro epithelial cell growth on titanium surfaces which had been instrumented by 3 types of periodontal curets. Commercially pure titanium discs were uniformly scaled using plastic, stainless steel, or gold-coated curets and then seeded with a microdot (50,000 cells per 10 microliters) of rat gingival epithelial cells. After 5 days of growth, epithelial cell surface area (mm2) coverage was measured on photographed specimens using a computer digitizing system. Scanning electron microscopic evaluation showed a denser central core of cellular growth on the stainless steel-treated surfaces as compared to the other surface treatments. Epithelial surface area coverage on the stainless steel, plastic, and control groups did not vary significantly among groups. However the gold-coated curet exposed surfaces supported significantly less epithelial growth than the stainless steel and control surfaces. A better understanding of in vitro epithelial cell responses to different titanium surface characteristics will assist in designing implant scaling procedures which allow maintenance of the implant perimucosal seal.

Quality of periodontal healing. III: Periodontal width following long epithelial junction or reparative cementum formation.

The purpose of the present study was to compare the width of the periodontal space following two different marginal periodontal healing reactions (long epithelial junction and connective tissue healing involving formation of reparative cementum) with the width of normal periodontium, either intact or following reattachment. Twelve permanent lateral incisors in monkeys from a previous experimental study on periodontal wound healing on denuded surfaces were selected based on strict healing criteria. The periodontal width was measured histomorphometrically and compared to the periodontal width on the contra lateral side of the same root where reattachment had taken place and with the width of normal periodontal space of neighbouring untreated teeth. The mean periodontal width for both after formation of a long junctional epithelium and reparative cementum were significantly larger than the control width, approximately 290%. This may be taken as evidence of an impaired functional adaptation of the reparative tissue compared to a reestablished or normal periodontal membrane.

In vitro growth of periodontal fibroblasts on treated cementum.

The aim of the present study was to assess the ability in vitro of phosphoric and citric acids, applied on human root cementum, to neutralize noxious plaque and calculus and to allow the growth of human gingival fibroblasts. Fibroblasts grown on cementum treated with phosphoric acid appeared typically elongated and aligned parallel to the root surface. Fibroblasts grown on cementum treated with citric acid, in both normal and periodontally diseased teeth, lost their elongated shape, acquiring polygonal borders with irregular cytoplasmic extrusions, and the cell density was significantly lower. These findings suggest that phosphoric acid cleaning of both normal and diseased root surfaces may result in an oriented, high rate of fibroblastic growth with more effective periodontal cellular proliferation than that observed after citric acid treatment.

Age-related development of the long-junctional epithelium in the senescence-accelerated mouse.

The mesial gingiva of the maxillary first molar of SAM-R/1/Iw (senescence-accelerated mouse, resistant/1/Iwate) and the P/2/Iw (prone/2/Iwate) were studied morphologically by quantitative and qualitative methods as a function of age (one-, two-, six-, 12-, and 16-month-old). In this region, neither plaque nor periodontitis has been found (Sashima et al., 1990a). Statistical analyses of the gingival tissues in this region in both strains showed that, with age, the length of the junctional epithelium (JED) increased, the depth of the gingival sulcus (GSD) did not change, and the height of the gingival crest (GCD) decreased. Therefore, long-junctional epithelium developed, and gingival recession occurred, since a periodontal pocket did not develop with age in either strain. In particular, there was a strong correlation between the JED and age in both strains (r = 0.84, p less than 0.05 for the R/1/Iw, and r = 0.92, p less than 0.05 for the P/2/Iw). The JED, GSD, and GCD of the P/2/Iw were higher than each of those in the R/1/Iw after six months of age; however, there were no significant differences between the R/1/Iw and the P/2/Iw for any age group (one-, two-, six-, 12-, and 16-month-old).

Formation of the periodontal attachment in developing rabbit permanent incisors.

Permanent incisors from fetal New Zealand white rabbits aged 25-30 days in utero and from 1-20 days postnatally were processed for light microscopy. Examination of longitudinal and cross-sections revealed that cementum deposition and fiber attachment first occurred at 27 days in utero. A tooth-to-bone course of principal fibers confined to lateral aspects of the teeth was observed in 29-day fetal incisors, whereas lingually the periodontal ligament remained undeveloped as late as 5 days postnatally. In 20-day postnatal incisors, the development of the functional periodontal ligament was complete as evidenced by the fiber arrangement on all aspects of the root-analogue tooth surface. This study shows that 25-26-day fetal rabbit permanent incisors represent the end of the follicular phase, whereas 27-day fetal to 20-day postnatal teeth demonstrate distinct histologic characteristics of initial, incomplete and complete periodontal fiber attachment.