Dental pulp stem cells, niches, and notch signaling in tooth injury.

Stem cells guarantee tissue repair and regeneration throughout life. The decision between cell self-renewal and differentiation is influenced by a specialized microenvironment called the 'stem cell niche'. In the tooth, stem cell niches are formed at specific anatomic locations of the dental pulp. The microenvironment of these niches regulates how dental pulp stem cell populations participate in tissue maintenance, repair, and regeneration. Signaling molecules such as Notch proteins are important regulators of stem cell function, with various capacities to induce proliferation or differentiation. Dental injuries often lead to odontoblast apoptosis, which triggers activation of dental pulp stem cells followed by their proliferation, migration, and differentiation into odontoblast-like cells, which elaborate a reparative dentin. Better knowledge of the regulation of dental pulp stem cells within their niches in pathological conditions will aid in the development of novel treatments for dental tissue repair and regeneration.

Timing of the expression of enamel gene products during mouse tooth development.

In order to understand the mechanisms involved in tooth development it is important to define the timing for tissue-specific gene expression. A consequence of ameloblast cell differentiation is the sequential expression of tissue-specific genes whose products form the enamel extracellular matrix. The ameloblast phenotype has been characterized as consisting of two major classes of proteins: amelogenins and non-amelogenin proteins such as anionic enamel proteins (enamelins, tuft proteins, tuftelin, sulfated proteins) and enamel proteases. The postulated functions for the anionic enamel proteins are as nucleators for hydroxyapatite crystal formation while amelogenins control the crystal size, growth and orientation. While the amelogenins have been well characterized, detailed knowledge for anionic enamel proteins has been sparse. In the present study, we designed experiments to characterize one of the anionic enamel proteins from mouse molars, tuftelin, and to determine the timing of expression of this protein during molar tooth development. Our results showed the initial detection of tuftelin transcripts within proliferating inner enamel epithelial cells at very early stages of tooth development (13 days of embryonic development equivalent to the bud stage of tooth development). These data provide direct evidence that invalidates previous dogmas that enamel proteins were synthesized by polarized, non-dividing, fully differentiated ameloblast cells. In addition, tuftelin was found to be synthesized also by dental papilla mesenchyme cells suggesting that this protein is not enamel-specific. These data taken together open the possibility that the tuftelin present in the dentino-enamel junction could be secreted by both, preodontoblast cells and preameloblast cells. It might also suggest a possible different role for tuftelin than nucleator of hydroxyapatite crystals.

Early dental epithelial transcription factors distinguish ameloblastoma from keratocystic odontogenic tumor.

The aim of the study was to characterize the molecular relationship between ameloblastoma and keratocystic odontogenic tumor (KCOT) by means of a genome-wide expression analysis. Total RNA from 27 fresh tumor samples of 15 solid/multicystic intraosseous ameloblastomas and 12 sporadic KCOTs was hybridized on Affymetrix whole genome arrays. Hierarchical clustering separated ameloblastomas and KCOTs into 2 distinct groups. The gene set enrichment analysis based on 303 dental genes showed a similar separation of ameloblastomas and KCOTs. Early dental epithelial markers PITX2, MSX2, DLX2, RUNX1, and ISL1 were differentially overexpressed in ameloblastoma, indicating its dental identity. Also, PTHLH, a hormone involved in tooth eruption and invasive growth, was one of the most differentially upregulated genes in ameloblastoma. The most differentially overexpressed genes in KCOT were squamous epithelial differentiation markers SPRR1A, KRTDAP, and KRT4, as well as DSG1, a component of desmosomal cell-cell junctions. Additonally, the epithelial stem cell marker SOX2 was significantly upregulated in KCOT when compared with ameloblastoma. Taken together, the gene expression profile of ameloblastoma reflects differentiation from dental lamina toward the cap/bell stage of tooth development, as indicated by dental epithelium-specific transcription factors. In contrast, gene expression of KCOT indicates differentiation toward keratinocytes.

Periodontal disease activity: a critical assessment.

Review of investigations using longitudinal probing and radiographic assessments indicate that the prevalence, magnitude, rate, and temporal patterns of periodontal destruction as well as the ability of clinical and laboratory tests to detect and predict loss of clinical attachment need further investigation. These unresolved issues are discussed and their impact on the practice of periodontics is examined.

Histometric evaluation of periodontal surgery. III. The effect of bone resection on the connective tissue attachment level.

The purpose of this study was to evaluate the effect of surgical elimination of the osseous walls of angular bony defects on the connective tissue attachment and alveolar bone levels. Using a Rhesus monkey model, 36 periodontal pockets in four animals were operated on while the contralateral pockets served as unoperated controls. Plaque control was maintained until the animals were killed 1 year after surgery. Following routine processing, the interdental tissues were analyzed histometrically. Comparing measurements of surgically treated and untreated sites, resection of the osseous walls of interdentally located angular bony defects caused not only a reduction in the height of the alveolar bone but also a significant loss of connective tissue attachment. The procedure also resulted in the elimination of angular bony defects, intrabony pockets, and in addition, reduced the height of the interdental soft tissue.

The attachment between tooth and gingival tissues after periodic root planing and soft tissue curettage.

Utilizing a nonhuman primate model, a study was carried out to determine the nature of the attachment between the tooth and the gingival tissues following periodic root planing and soft tissue curettage. Under the conditions of this investigation, periodic root planing and soft tissue curettage combined with thrice weekly plaque control resulted in the formation of a long junctional epithelium with no new connective tissue attachment. In eight of the 22 experimental pockets, however, this procedure produced discontinuities or "windows" in the junctional epithelium. The coronal attachment of gingival tissues to the root surface (increased resistance to probing) commonly reported following root planing and soft tissue curettage appears to result from the formation of a long junctional epithelium rather than new connective tissue attachment.

Histologic comparisons of interproximal gingival tissues related to the presence or absence of bleeding.

Interproximal gingival tissues were compared histologically relative to the presence or absence of bleeding after stimulation with a wooden interdental cleaner. Fifteen bleeding and 15 nonbleeding interproximal gingival biopsy specimens were obtained from 30 patients and processed for light microscopic evaluation. Morphometric analysis of tissue components revealed that bleeding areas had a significantly greater per cent of inflamed connective tissue. The inflammatory lesion in the bleeding specimens was primarily in the midinterproximal area. From a diagnostic standpoint, this information provides a rationale for the use of bleeding to detect inflammatory lesions in the midinterproximal region.

Primate model for testing periodontal treatment procedures: I. Histologic investigation of localized periodontal pockets produced by orthodontic elastics.

Experimental periodontal lesions have been produced in Rhesus monkeys using orthodontic elastics. These lesions are characterized by an irreversible apical positioning of the junctional epithelium and both horizontal and angular bone loss. It was concluded that this lesion is suitable for testing the effect of periodontal treatment.

Synthetic bioabsorbable barrier for regeneration in human periodontal defects.

Guided tissue regeneration (GTR) may result in the formation of new bone, cementum, and periodontal ligament. The purpose of this study was to assess the efficacy of a resorbable synthetic material, which has been used extensively in general surgery for wound support, to promote GTR. Forty healthy patients with adult periodontitis, each having a Class II furcation defect, participated in the study. After initial therapy, mucoperiosteal flaps were elevated and furcations debrided with hand and rotary instruments. In 20 patients the molar Class II furcation defects were treated with a GTR procedure using the resorbable synthetic material (experimental), and 20 patients received a mucoperiosteal flap debridement procedure without barrier placement (control). Probing depth and attachment level measurements were taken immediately before surgery, at 6 weeks, and 2, 3, 4, 5, and 6 months after surgery. All areas healed uneventfully. Comparison of clinical attachment level measurements indicated significantly greater gain of attachment at sites receiving barriers. Fifteen of 20 Class II furcations in the synthetic barrier group, but only one of 20 in the control group, were converted to Class I defects. Barriers were still clinically detectable at 4 weeks, but were absent at 6 weeks. The synthetic barriers enhanced gain of clinical attachment in human Class II furcation defects.

Maintenance of healed periodontal pockets after a single episode of root planing.

The present study evaluated the clinical stability of healed periodontal pockets over a 3 month time period to determine whether this time interval is appropriate for periodontal maintenance therapy. The clinical characteristics of 128 pockets (3-7 mm depth) distributed in 10 patients, were monitored immediately before and 4, 8 and 16 weeks after a single episode of subgingival root planing. The clinical parameters measured were: pocket depth and bleeding after probing with 15, 25 and 50 gm and manual probing, gingival margin location, clinical attachment level, and gingival and plaque indices. Oral hygiene instruction and supragingival cleaning were given at each time point. An average aggregated score for each subject and for each parameter was calculated at each time point. A repeated measure all within analysis of variance was done, and the Tukey multiple range test was used to assess the significance of differences among and between the means. The significant decreases in plaque, gingival and bleeding indices, and pocket depth as well as the significant gingival recession and gain of clinical attachment which were present at the 4 week point were maintained at 8 and 16 weeks after root planing. It was concluded that the favorable clinical changes which occur in periodontal pockets within 1 month after a single episode of subgingival root planing combined with improved oral hygiene can be maintained for an additional 3 month time period.

Initial healing of periodontal pockets after a single episode of root planing monitored by controlled probing forces.

THE PURPOSE OF the present study was to evaluate, using controlled probing forces, the response of periodontal pockets to a single episode of root planing. The clinical characteristics of 128 pockets (3-7 mm depth) distributed in ten subjects, were monitored immediately before and 1, 2, 3, and 4 weeks after a single episode of subgingival root planing. The clinical parameters measured were: pocket depth and bleeding after probing with 15 gm, 25 gm, 50 gm of controlled probing force and manual probing, gingival margin location, loss of attachment, gingival and plaque indices. Oral hygiene instruction and supragingival prophylaxis were given at each time point. An average aggregated score for each subject for each parameter was calculated at each time point. A repeated measure all-within analysis of variance was done, and the Tukey multiple range test was used to assess the significance of differences among and between the means. Plaque and gingival indices decreased significantly after 1 week. Significant pocket depth reduction (initial) occurred 1 week after root planing, and reduced further (secondary) at 3 weeks. Initial pocket reduction was associated with significant gingival recession, whereas secondary pocket reduction was associated with significant gain of clinical attachment. Bleeding upon probing was virtually absent after 3 weeks. All probing changes were detected more consistently using controlled insertion pressures. It was concluded that substantial reduction in pocket depth occurs within 3 weeks after a single episode of root planing owing to initial gingival recession and secondary gain in clinical attachment.

Factors influencing periodontal repair and regeneration.

The purpose of the present study was to evaluate independently the regeneration capacity of a reduced periodontium, and the potential for new connective tissue attachment to a periodontally exposed root surface. A reduced periodontium was produced around a maxillary central incisor in two Rhesus monkeys using orthodontic elastics. The regeneration capacity of this reduced peridontium was investigated by extracting the periodontally involved tooth and transplanting into the socket the contralateral incisor which had a normal periodontium. The potential for new connective tissue attachment to a periodontally exposed root surface was evaluated by transplanting an exposed root into a normal periodontium. These exposed roots were the incisors extracted from the reduced peridontium. Control specimens were obtained by extracting and reimplanting or transplanting the remaining maxillary and mandibular incisors. Forty days after the experimental procedures, block sections were removed and prepared for histologic analysis. The exposed root surface that had been put into the normal periodontium was lined with epithelium interposed between root surface and alveolar bone. The normal root surface that had been put into the reduced periodontium had connective tissue reattachment in the periodontal ligament and supracrestal regions. Areas of ankylosis and root resorption were present in all specimens, as was new cementum in the periodontal ligament region. The results indicated that root surface alterations, rather than presence of a reduced periodontium, inhibited new connective tissue attachment.

Primate model for testing periodontal treatment procedures: II. Production of contralaterally similar lesions.

Experimental periodontal pockets were produced bilaterally in four Rhesus monkeys. The pockets were analyzed histometrically and the resultant data were subjected to statistical analysis. There were but few significant differences between pockets on contralateral tooth surfaces, and in each case, the magnitudes of the observed differences were so small as to be of no biological significance. The use of this model system for future studies on periodontal treatment was discussed. It was shown that by utilizing only two monkeys, the model system can discern treatment effects on the measurable parameters of as little as 0.3 mm.

Cell populations associated with conversion from bleeding to nonbleeding gingiva.

The purpose of this study was to observe changes in cell populations of the interdental gingival tissue, which accompanied the conversion of a bleeding to a nonbleeding state induced by scaling and improved oral hygiene. Fifteen bleeding and 18 stopped-bleeding interproximal gingival biopsies were obtained from 33 patients and processed for light microscopic evaluation. The morphometric analysis of eight connective tissue components revealed that the percentage volume density of all inflammatory cells decreased, and the percentage of fibroblasts and collagen increased, when the gingiva changed from a bleeding to a nonbleeding state. Furthermore, the inflammatory cell infiltrate in bleeding and stopped-bleeding specimens was dominated by mononuclear cells of the lymphocyte/macrophage/monocyte group, while plasma cells and polymorphonuclear leukocytes comprised only a small fraction of the inflammatory cells present. Significant repair of gingival connective tissue had occurred in the stopped-bleeding specimens.

Effect of personal oral hygiene on bleeding interdental gingiva. Histologic changes.

A previous study demonstrated that the combination of subgingival scaling and improved oral hygiene resulted in a reduction of clinical and histological signs of interdental gingival inflammation, changes that were associated with a cessation of interdental gingival bleeding. The present study compared, histologically, the interdental tissues of bleeding sites with sites that initially bled but had been converted to nonbleeding by an oral hygiene program alone. Morphometric analysis of interdental gingiva demonstrated that conversion from bleeding to nonbleeding was associated with a significant reduction in the inflamed connective tissue component. This study showed that an oral hygiene program consisting of toothbrushing and interdental cleaning could significantly reduce interdental inflammation, and that bleeding determinations monitored the effects of this therapy.

Effects of personal oral hygiene and subgingival scaling on bleeding interdental gingiva.

The aim of this study was to determine the effects of personal oral hygiene and subgingival scaling on bleeding interdental gingiva. The Eastman Interdental Bleeding Index (EIBI) was used to clinically evaluate interdental gingival status. Forty-seven bleeding interdental sites in 47 patients were divided into three groups. Sites in Group I bled on stimulation with wooden interdental cleaners. Groups II and III initially bled but were converted to nonbleeding with oral hygiene alone or oral hygiene combined with subgingival scaling, respectively. Interdental gingival biopsies were obtained and subjected to morphometric analysis to compare the three groups. The findings from this study indicated that: (A) personal oral hygiene reduced the magnitude and extent of the interdental inflammatory lesion; (B) subgingival scaling plus oral hygiene decreased the interdental inflammatory lesion to a greater extent than oral hygiene alone; (C) significant repair of the interdental lesion occurred within four weeks; and (D) the EIBI was an effective method for monitoring the effects of therapy directed towards resolution of the interdental inflammatory lesion.

Histologic characteristics associated with bleeding after probing and visual signs of inflammation.

This study was designed to characterize histologically the gingival lesion associated with visual signs of inflammation and bleeding after probing. Sixty midfacial gingival sites in 26 persons were evaluated for visual presence of absence of inflammation and bleeding after probing using a controlled insertion pressure of 25 gm. In order to delineate the area for histologic analysis, a reference incision was made on the facial surface of the gingiva which corresponded to the depth and mesio-distal extent of the area probed and evaluated for signs of inflammation. Gingival biopsy specimens were morphometrically analyzed to determine the percentages of cell rich--collagen poor connective tissue and blood vessel lumens. Histometric data were grouped and compared with respect to the presence or absence of inflammation and bleeding. Specimens associated visually with inflammation had significantly greater percentages of both cell rich--collagen poor connective tissue and blood vessel lumens. Bleeding upon probing was associated with a significantly greater percentage of cell rich--collagen poor connective tissue without an increase of blood vessel lumens. It was concluded that both observed signs of inflammation and bleeding after probing can be used to detect inflammatory lesions in the gingiva. However, in areas inaccessible for evaluation of inflammation, bleeding determinations using controlled insertion pressures provided an objective diagnostic method for detecting the presence of an inflammatory lesion.

Depth of periodontal probe penetration related to clinical and histologic signs of gingival inflammation.

The purpose of this study was to determine if the depth of periodontal probe penetration into the gingival sulcus was related to bleeding after probing with a standardized force, visual signs of gingival inflammation, and histological parameters of gingival inflammation. Sixty midfacial gingival sites in 26 persons were evaluated for presence or absence of visible inflammation and bleeding after probing using a controlled insertion pressure of 25 gm. In order to delineate the area for histologic analysis, a reference incision was made on the facial surface of the gingiva which corresponded to the depth and mesio-distal extent of the area probed and evaluated for visual signs of inflammation. Gingival biopsy specimens were histometrically analyzed to determine the depth of probe penetration and percentage of inflamed connective tissue. Statistical analysis of the data demonstrated that probe penetration was significantly greater in the presence of visible inflammation, but not with bleeding after probing. Furthermore, only a weak correlation existed between depth of probe penetration and percentage of inflamed connective tissue adjacent to the area probed.

Relationships between epithelium and connective tissue in inflamed gingiva.

Since chronic inflammation has been associated with degenerative and proliferative responses in connective tissue and epithelium, it was decided to investigate quantitatively the proportions of epithelium and connective tissue in inflamed gingiva. Forty-five midfacial gingival sites were assessed prior to biopsy for the presence or absence of bleeding after probing using a controlled insertion force fo 25 g. In order to delineate the corresponding area for histologic analysis, a reference incision was made on the facial surface of the gingiva which demarcated the depth and mesio-distal extent of the area probed. Step-serial bucco-lingual sections were morphometrically analyzed to determine the percentages of epithelium, inflamed, and noninflamed connective tissues coronal to the reference incision. The data was compared with respect to the presence or absence of bleeding after probing. Specimens which bled after probing had significantly less epithelium and more connective tissue. The increase in overall connective tissue was due to a significant increase in the inflamed component, and it appeared that the decrease in epithelium corresponded to the increase in inflamed connective tissue. The findings of diminished epithelium overlying inflamed connective tissue reinforces the concept that connective tissue status is important for determining structure and function of epithelium.

Interproximal gingival inflammation related to the conversion of a bleeding to a nonbleeding state.

Elimination of gingival bleeding has been related to a reduction in inflammation; however, histologic data are not available to support this association. The purpose of this study was to characterize the histology of interproximal gingiva that was converted from a bleeding to a nonbleeding state. An interproximal gingival biopsy was obtained from each of 32 patients, 15 of whom bled upon stimulation with a soft wooden interdental cleaner. The remaining 17 biopsies were obtained from sites which initially bled, but were converted to nonbleeding by scaling and interproximal plaque control. Specimens were processed for light microscopic evaluation and subjected to a morphometric analysis for various tissue components. Data from bleeding and "stopped bleeding" specimens were compared using analysis of covariance. The results indicated that "stopped bleeding" specimens had significantly less inflamed connective tissue. It was concluded that the conversion of a bleeding to a nonbleeding state corresponds with a histological reduction in the magnitude of the interproximal inflammatory lesion and provides a rationale, therefore, for the use of bleeding to monitor the effects of therapeutic methods.