Translational research and therapeutic applications of neural crest-derived stem cells in regenerative periodontology

Regeneration of periodontal tissues aims to utilize tissue engineering techniques to restore lost periodontal tissues including the cementum, periodontal ligament and alveolar bone. Regenerative dentistry and its special field regenerative periodontology represent relatively new and emerging branches of translational stem cell biology and regenerative medicine focusing on replacing and regenerating dental tissues to restore or re-establish their normal function lost during degenerative diseases or acute lesions. The regeneration itself can be achieved through transplantation of autologous or allogenic stem cells, or by improving the tissue self-repair mechanisms (e.g. by application of growth factors). In addition, a combination of stem cells or stem cell-containing tissue with bone implants can be used to improve tissue integration and the clinical outcome. As the oral cavity represents a complex system consisting of teeth, bone, soft tissues and sensory nerves, regenerative periodontology relies on the use of stem cells with relatively high developmental potential. Notably, the potential use of pluripotent stem cell types such as human embryonic stem cells or induced pluripotent stem cells is still aggravated by ethical and practical problems. Thus, other cellular sources such as those readily available in the postnatal craniofacial area and particularly in oral structures offer a much better and realistic alternative as cellular regenerative sources. In this review, we summarize current knowledge on the oral neural crest-derived stem cell populations (oNCSCs) and discuss their potential in regenerative periodontology.

Revascularization and periapical repair after endodontic treatment using apical negative pressure irrigation versus conventional irrigation plus triantibiotic intracanal dressing in dogs` teeth with apical periodontitis

Objective. The objective of this study was to evaluate in vivo the revascularization and the apical and periapical repair after endodontic treatment using 2 techniques for root canal disinfection (apical negative pressure irrigation versus apical positive pressure irrigation plus triantibiotic intracanal dressing) in immature dogs` teeth with apical periodontitis. Study design. Two test groups of canals with experimentally induced apical periodontitis were evaluated according to the disinfection technique: Group 1, apical negative pressure irrigation (EndoVac system), and Group 2, apical positive pressure irrigation (conventional irrigation) plus triantibiotic intracanal dressing. In Group 3 (positive control), periapical lesions were induced, but no endodontic treatment was done. Group 4 (negative control) was composed of sound teeth. The animals were killed after 90 days and the maxillas and mandibles were subjected to histological processing. The sections were stained with hematoxylin and eosin and Mallory Trichrome and examined under light microscopy. A description of the apical and periapical features was done and scores were attributed to the following histopathological parameters: newly formed mineralized apical tissue, periapical inflammatory infiltrate, apical periodontal ligament thickness, dentin resorption, and bone tissue resorption. Intergroup comparisons were done by the Kruskal-Wallis and Dunn`s tests (alpha = 0.05). Results. Although statistically significant difference was found only for the inflammatory infiltrate (P < .05), Group 1 presented more exuberant mineralized formations, more structured apical and periapical connective tissue, and a more advanced repair process than Group 2. Conclusion. From the histological observations, sodium hypochlorite irrigation with the EndoVac system can be considered as a promising disinfection protocol in immature teeth with apical periodontitis, suggesting that the use of intracanal antibiotics might not be necessary. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010; 109: 779-787)

Matrix Metalloproteinase Expression in Teeth with Apical Periodontitis Is Differentially Modulated by the Modality of Root Canal Treatment

Introduction: The objective of this study was to investigate the expression of matrix metalloproteinases (MM Ps) in apical periodontitis and during the periapical healing phase after root canal treatment. Methods: Apical periodontitis was induced in dog teeth, and root canal treatment was performed in a single visit or by using an additional calcium hydroxide root canal dressing. One hundred eighty days after treatment the presence of inflammation was examined, and tissues were stained to detect bacteria. Bacterial status was correlated to the degree of tissue organization, and to further investigate molecules involved in this process, tissues were stained for MMP-1, MMP-2, MMP-8, and MMP-9. Data were analyzed by using one-way analysis of variance followed by Tukey test or Kruskal-Wallis followed by Dunn test. Results: Teeth with apical periodontitis that had root canal therapy performed in a single visit presented an intense inflammatory cell infiltrate. Periapical tissue was extremely disorganized, and this was correlated with the presence of bacteria. Higher MMP expression was evident, similar to teeth with untreated apical periodontitis. In contrast, teeth with apical periodontitis submitted to root canal treatment with calcium hydroxide presented a lower inflammatory cell infiltrate. This group had moderately organized connective tissue, lower prevalence of bacteria, and lower number of MMP-positive cells, similar to healthy teeth submitted to treatment. Conclusions: Teeth treated with calcium hydroxide root canal dressing exhibited a lower percentage of bacterial contamination, a lower MMP expression, and a more organized extracellular matrix, unlike those treated in a single visit. This suggests that calcium hydroxide might be beneficial in tissue repair processes. (J Endod 2010;36:231-237)

High Matrix Metalloproteinase Activity Is a Hallmark of Periapical Granulomas

Introduction: The inability to distinguish periapical cysts from granulomas before performing root canal treatment leads to uncertainty in treatment outcomes because cysts have lower healing rates. Searching for differential expression of molecules within cysts or granulomas could provide information with regard to the identity of the lesion or suggest mechanistic differences that may form the basis for future therapeutic intervention. Thus, we investigated whether granulomas and cysts exhibit differential expression of extracellular matrix (ECM) molecules. Methods: Human periapical granulomas, periapical cysts, and healthy periodontal ligament tissues were used to investigate the differential expression of ECM molecules by microarray analysis. Because matrix metalloproteinases (MMP) showed the highest differential expression in the microarray analysis, MMPs were further examined by in situ zymography and immunohistochemistry. Data were analyzed by using one-way analysis of variance followed by the Tu-key test. Results: We observed that cysts and granulomas differentially expressed several ECM molecules, especially those from the MMP family. Compared with cysts, granulomas exhibited higher MMP enzymatic activity in areas stained for MMP-9. These areas were composed of polymorphonuclear cells (PMNs) in contrast to cysts. Similarly, MMP-13 was expressed by a greater number of cells in granulomas compared with cysts. Conclusion: Our findings indicate that high enzymatic MIMP activity in PMNs together with MMP-9 and MMP-13 stained cells could be a molecular signature of granulomas unlike periapical cysts. (J Endod 2009;35:1234-1242)

TNF-alpha Promotes an Odontoblastic Phenotype in Dental Pulp Cells

Dental pulp cells can differentiate toward an odontoblastic phenotype to produce reparative dentin beneath caries lesions. However, the mechanisms involved in pulp cell differentiation under pro-inflammatory stimuli have not been well-explored. Thus, we hypothesized that the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) could be a mediator involved in dental pulp cell differentiation toward an odontoblastic phenotype. We observed that TNF-alpha-challenged pulp cells exhibited increased mineralization and early and increased expression of dentin phosphoprotein (DPP), dentin sialoprotein (DSP), dentin matrix protein-1, and osteocalcin during a phase of reduced matrix metalloproteinase (MMP) expression. We investigated whether these events were related and found that p38, a mitogen-activated protein kinase, differentially regulated MMP-1 and DSP/DPP expression and mediated mineralization upon TNF-alpha treatment. These findings indicate that TNF-alpha stimulates differentiation of dental pulp cells toward an odontoblastic phenotype via p38, while negatively regulating MMP-1 expression.

SEM Study of Apical Morphological Alterations in Primary Teeth with Vital and Necrotic Pulps

This study evaluated, by SEM, the morphology of human primary teeth roots. Twenty-four teeth were divided into 3 groups: pulp vitality (group I) and pulp necrosis without (group II) and with apical periodontitis (group III). Roots were analyzed by the presence of periodontal ligament (PDL) fibers and resorption areas. In groups I and II, presence of PDL fibers and absence of resorption were observed in all cases (100%), while all specimens (100%) of group III showed no PDL fibers and resorption areas. In conclusion, there are morphological differences in the apical region of primary teeth with different pulpal and periapical pathologies.

A comparison study of periapical repair in dogs` teeth using RoekoSeal and AH plus root canal sealers: A histopathological evaluation

The objective of the study was to evaluate the biocompatibility of RoekoSeal sealer Roeko (Dental Products, Langenau, Germany) with the periapical tissues of dogs and compare it with AH Plus sealer (Dentsply/De Trey, Konstanz, Germany). The pulps of 32 root canals were removed, the apical cementum layer perforated, the biomechanical preparation performed, and the root canals filled by lateral condensation technique. Ninety days after the surgery, the animals were euthanized, the bone with teeth removed, and the samples prepared for histopathological analysis. In group 1 (RoekoSeal Automix), deposition of mineralized tissue was observed, with complete newly mineralized apical formed tissue in 43.8% and partial sealing in 56.2%. In group 2 (AH Plus), in 12.5% there was complete newly mineralized apical formed tissue, in 75% the sealing was partial, and in 12.5% there was no sealing (p < 0.05). There were no differences between the groups in relation to the inflammatory infiltrate; thickness of the periodontal ligament; and the resorption of dentin, cementum or bone (p > 0.05).

Myeloperoxidase activity is increased in gingival crevicular fluid and whole saliva after fixed orthodontic appliance activation

Introduction: Orthodontic tooth movement uses mechanical forces that result in inflammation in the first days. Myeloperoxidase (MPO) is an enzyme found in polymorphonuclear neutrophil (PMN) granules, and it is used to estimate the number of PMN granules in tissues. So far, MPO has not been used to study the inflammatory alterations after the application of orthodontic tooth movement forces. The aim of this study was to determine MPO activity in the gingival crevicular fluid (GCF) and saliva (whole stimulated saliva) of orthodontic patients at different time points after fixed appliance activation. Methods: MPO was determined in the GCF and collected by means of periopaper from the saliva of 14 patients with orthodontic fixed appliances. GCF and saliva samples were collected at baseline, 2 hours, and 7 and 14 days after application of the orthodontic force. Results: Mean MPO activity was increased in both the GCF and saliva of orthodontic patients at 2 hours after appliance activation (P<0.02 for all comparisons). At 2 hours, PMN infiltration into the periodontal ligament from the orthodontic force probably results in the increased MPO level observed at this time point. Conclusions: MPO might be a good marker to assess inflammation in orthodontic movement; it deserves further studies in orthodontic therapy. (Am J Orthod Dentofacial Orthop 2010;138:613-6)

Histologic Evaluation of the Buccal and Lingual Bone Plates in Anterior Dog Teeth: Possible Influence on Implant Dentistry

Background: Recent studies in animals have shown pronounced resorption of buccal bone plate after immediate implantation. The sectioning of experimental material for histologic evaluation of the bone plates could provide valuable information about the possible effect of bone exposure in periodontal and implant surgeries. Methods: Twenty-four incisors were collected from dogs. After decalcification, the blocks were immersed in paraffin and bucco-lingual histologic sections were examined under light microscope. Some sections were reserved for immunohistochemical analysis. Results: The bone density, the width of the bone plates, and the percentage of vessels presented in the periodontal ligament and periosteum were analyzed in the buccal and lingual bone plates, which were divided corono-apically into thirds. The buccal bone plates showed statistically higher bone density compared to the lingual bone plates in the coronal thirds. The width of both bone plates increased from the coronal to the apical third, but all the buccal thirds were significantly thinner compared to the lingual thirds. No statistically significant differences were found between the bone plates for the percentage of area occupied by the blood vessels in the periodontal ligament or periosteum. Conclusion: It is reasonable to conclude that the higher bone density, represented by the lower number of marrow spaces, in association with the thinner aspect of the buccal bone plates made them more fragile to absorb compared to the lingual bone plates, especially during mucoperiosteal procedures. J Periodontol 2017;82:872-877.

Influence of Post and Resin Cement on Stress Distribution of Maxillary Central Incisors Restored with Direct Resin Composite

The current study evaluated the influence of two endodontic post systems and the elastic modulus and film thickness of resin cement on stress distribution in a maxillary central incisor (MCI) restored with direct resin composite using finite element analysis (FEA). A three-dimensional model of an MCI with a coronary fracture and supporting structures was performed. A static chewing pressure of 2.16 N/mm(2) was applied to two areas on the palatal surface of the composite restoration. Zirconia ceramic (ZC) and glass fiber (GF) posts were considered. The stress distribution was analyzed in the post, dentin and cement layer when ZC and GF posts were fixed to the root canals using resin cements of different elastic moduli (7.0 and 18.6 GPa) and different layer thicknesses (70 and 200 mu m). The different post materials presented a significant influence on stress distribution with lesser stress concentration when using the GF post. The higher elastic modulus cement created higher stress levels within itself. The cement thicknesses did not present significant changes.

Information for the diagnosis and treatment of root resorption due to tooth replantation

A favorable prognosis after tooth avulsion depends on some variables, such as the extra-alveolar period and storage medium. Vitality of the periodontal ligament cells is considered a critical factor for a successful outcome without root resorption. The dental surgeon is provided with clinical information and radiographic findings to establish a diagnosis and may rely on current available guidelines. Once trauma has occurred, treatment must be quick and effective, and periodic follow-up must be performed. Clinical, radiographic, and histologic characteristics for each type of root resorption due to tooth replantation are presented, with the aim to provide information for the diagnosis and treatment of healing complications.

Short-term vs long-term calcium hydroxide therapy after immediate tooth replantation: a histomorphometric study in monkey's teeth

Endodontic treatment is an important step of tooth replantation protocols, but the ideal moment for definitive obturation of replanted teeth has not yet been established. In this study, a histomorphometric analysis was undertaken to evaluate the repair process on immediate replantation of monkeys teeth after calcium hydroxide (CH) therapy for 1 and 6 months followed by root canal filling with a CH-based sealer (Sealapex (R)). The maxillary and mandibular lateral incisors of five female Cebus apella monkeys were extracted, kept in sterile saline for 15 min, replanted and splinted with stainless steel orthodontic wire and composite resin for 10 days. In Group I (control), definitive root canal filling was performed before tooth extraction. In Groups II and III, CH therapy started after removal of splint, and definitive root canal filling was performed 1 and 6 months later, respectively. The animals were euthanized 9 months after replantation, and specimens were processed for histomorphometric analysis. In all groups, epithelial attachment occurred at the cementoenamel junction or very close to this region; the areas of resorption on root surface had small extension and depth and were repaired by newly formed cementum; and the periodontal ligament was organized. Statistical analysis of the scores obtained for the histomorphometric parameters did not show any statistically significant difference (P = 0.1221) among the groups. The results suggests that when endodontic treatment is initiated 10 days after immediate replantation and an antibiotic regimen is associated, definitive root canal filling can be performed after a short-term CH therapy.

Microscopic evaluation of induced tooth movement in traumatized teeth: an experimental study in rats

The clinical management of orthodontic patients with dental trauma before or during the treatment is mainly founded on clinical experience, expert opinions, and individual case reports. It is proposed in the literature that teeth sustaining mild trauma with minor damage to the periodontium (e.g. subluxation) should be followed for a period of time before being subjected to orthodontic forces. A minimum period of 3 months has been proposed. In this study, we used an animal model to investigate whether shorter observation periods could be established in case of mild trauma. The periradicular region of rat molars was examined microscopically to determine the biological events of tooth movement started 15 and 30 days after intentional subluxation using an experimental method to induce dentoalveolar trauma. Thirty adult male Wistar rats were assigned to 6 groups (n = 5): Group 1 (control no trauma/orthodontic movement); Group 2: the animals received an orthodontic device and were sacrificed after 7 days; Groups 3 and 4: dentoalveolar trauma (subluxation) was experimentally induced by the application of an axial force of 900 cN on the occlusal surface of the maxillary right first molar, and the animals were sacrificed after 22 and 37 days, respectively; and Groups 5 and 6: 15 and 30 days, respectively, after force application, an orthodontic device was installed and the rats were sacrificed 7 days later. In G5 and G6, the periodontal ligament and pulp tissue were rich in cellular elements and blood vessels, the alveolar bone was preserved, and the root surface presented only very small areas of surface resorption (cementum), maintaining the characteristics of normality. In conclusion, the microscopic alterations in the gingival and periodontal tissues in response to an experimentally induced mild dentoalveolar trauma simulating subluxation were not sufficient to contraindicate starting the orthodontic movement 15 and 30 days after trauma.

Tooth replantation after use of euro-coffins solution or bovine milk as storage medium: a histomorphometric analysis in dogs

Purpose: Euro-Collins solution was developed for the preservation of organs for transplantation, whose characteristics have raised interest for its use as a storage medium for avulsed teeth before replantation. This study evaluated histologically and morphometrically the healing process of dog teeth replanted after storage in Euro-Collins solution or bovine milk.Materials and Methods: Eighty roots of 4 young adult mongrel clogs were randomly assigned to 4 groups (n = 20) and the root canals were instrumented and obturated with gutta-percha and a calcium hydroxide-based sealer. After 2 weeks, the teeth were extracted and subjected to the following protocols: GI (negative control), replantation immediately after extraction; GII (positive control), bench-drying for 2 hours before replantation; GIII and GIV, immersion in 10 mL of whole bovine milk and Euro-Collins solution at 4 C, respectively, for 8 hours before replantation. The animals were sacrificed 90 days postoperatively. The pieces containing the replanted teeth were subjected to routine processing for histologic and histometric analyses under light microscopy and polarized light microscopy.Results: Root resorption was observed in all groups. GII exhibited the greatest loss of dental structure (P < .01), and inflammatory resorption was predominant in this group. Storage in milk showed poorer results than immediate replantation and storage in Euro-Collins solution (P < .01). The teeth stored in Euro-Collins solution presented similar extension of root resorption and periodontal ligament reorganization to those of immediately replanted teeth.Conclusions: The findings of this study suggest that the Euro-Collins solution is an adequate storage medium for keeping avulsed teeth for up to 8 hours before replantation.

Calcium Hydroxide Mixed With Camphoric p-Monochlorophenol or Chlorhexidine in Delayed Tooth Replantation

This study evaluated the repair process after delayed replantation of rat teeth, using calcium hydroxide (Ca(OH)(2)) mixed with camphorated p-monochlorophenol (CMCP), chlorhexidine 2% (CHX), or saline as temporary root canal dressing to prevent and/or control inflammatory radicular resorption. Thirty Wistar rats (Rattus norvegicus albinos) had their right upper incisor extracted, which was bench-dried for 60 minutes. The dental papilla, the enamel organ, the dental pulp, and the periodontal ligament were removed. The teeth were immersed in 2% acidulated-phosphate sodium fluoride solution for 10 minutes. The root canals were dried with absorbent paper cones and divided into 3 groups of 10 animals according to root canal dressing used: group 1: Ca(OH)(2) + saline, group 2: Ca(OH)(2) + CMCP, and group 3: Ca(OH)(2) + CHX 2%. Before replanting, the teeth sockets were irrigated with saline. Histological analysis revealed the presence of inflammatory resorption, replacement resorption, and ankylosis in all 3 groups. Statistical analysis showed a significant difference between group 3 and the other groups. The use of Ca(OH)(2) mixed with CMCP or CHX did not show an advantage over the use of Ca(OH)(2) mixed with saline in preventing and/or controlling inflammatory resorption in delayed replantation of rat teeth.