From biofilm disinfection to bacterial stem cell modulation and dentin matrix-driven stem cell mineralization

Caries or trauma are common occurrences in the permanent dentition of young patients. They often result in pulp necrosis, root canal infection, inflammation (apical periodontitis) and arrest of further root development. The consequences for the young patients can be overwhelming, ranging from intense pain to tooth loss. Currently, biological-based approaches exploiting patients’ own stem cells (revitalization treatment) are becoming more popular. They predictably achieve resolution of pain and inflammation and often succeed to promote further root development. Despite the favorable outcomes, the predictability of revitalization treatment has been questioned. Bacterial biofilms, the nature of stem cells and the quality and quantity of the bioactive stem cell guiding molecules released from the root canal are factors that may influence the treatment outcome.In the first part of this thesis, we investigated the anti-biofilm capacity of several root canal irrigants used during revitalization treatment. For the first time, we demonstrated that biofilm structure itself affects the action of the irrigants against biofilms. Next, we provided evidence that the balance between the unfavorable inflammatory and the favorable mineralizing stem cell phenotype can be regulated by specific cell surface receptors (TLR2) activated by bacterial and biofilm components. In the final part, we explored the mineralization capacity of several stem cell populations eligible for revitalization treatment, under the guidance of bioactive molecules derived from tooth dentin. We believe that the findings presented in this thesis could contribute in the optimization of revitalization clinical treatment protocols. That could increase treatment success and predictability

Effect of dentin matrix components on the mineralization of human mesenchymal stromal cells

In teeth with an injured pulp, dentin matrix orchestrates hard tissue repair through the release of dentin extracellular matrix components (dEMCs). dEMCs regulate the differentiation of resident mesenchymal stromal cells (MSCs), thereby affecting mineral deposition. In this study, we show that low-concentration solubilized dEMCs in osteogenic cultures of human umbilical cord mesenchymal stromal cells (UC-MSCs) and dental pulp stromal cells (DPSCs) enhanced mineral deposition, while adipose stromal cells (ASCs) were barely affected. Interestingly, UC-MSCs displayed significantly greater hydroxyapatite formation compared with DPSCs. UC-MSCs and DPSCs showed a dose-dependent viability and proliferation, whereas proliferation of ASCs remained unaffected. Qualitative analysis of the dEMC-supplemented osteogenic cultures through scanning electron microscopy demonstrated differences in the architecture of the deposited mineralized structures. Large-sized mineral accretions on a poorly organized collagen network were the prominent feature of UC-MSC cultures, while mineral nodules interspersed throughout a collagen mesh were observed in the respective DPSC cultures. The ability of dEMCs to induce mineralization varies between different human MSC types in terms of total mineral formation and architecture. Mineral formation by UC-MSCs exposed to low-concentration dEMCs proved to be the most efficient and therefore could be considered a promising combination for mineralized tissue engineering. Impact Statement This research has been conducted with the aim to contribute to the development of treatment modalities for the reconstruction of lost/damaged mineralized tissues. Currently, determining the most appropriate stromal cell population and signaling cues stands at the core of developing effective treatments. We provide new insights into the effect of innate inductive cues found in human dentin matrix components, on the osteogenic differentiation of various human stromal cell types. The effects of dentin extracellular matrix components on umbilical cord mesenchymal stromal cells have not been investigated before. The findings of this study could underpin translational research based on the development of techniques for mineralized tissue engineering and will be of great interest for the readership of Tissue Engineering Part A

The response of dual-species bacterial biofilm to 2% and 5% NaOCl mixed with etidronic acid:A laboratory real-time evaluation using optical coherence tomography

Aim: The addition of etidronic acid (HEDP) to sodium hypochlorite (NaOCl) could increase the antibiofilm potency of the irrigant, whilst maintaining the benefits of continuous chelation. Studies conducted so far have shown that mixing HEDP with NaOCl solutions of relatively low concentration does not compromise the antibiofilm efficacy of the irrigant. However, the working lifespan of NaOCl may decrease resulting in a reduction of its antibiofilm efficacy over time (efficiency). In this regard, continuous irrigant replenishment needs to be examined. This study investigated the response of a dual-species biofilm when challenged with 2% and 5% NaOCl mixed with HEDP for a prolonged timespan and under steady laminar flow. Methodology: Dual-species biofilms comprised of Streptococcus oralis J22 and Actinomyces naeslundii T14V-J1 were grown on human dentine discs in a constant depth film fermenter (CDFF) for 96 h. Biofilms were treated with 2% and 5% NaOCl, alone or mixed with HEDP. Irrigants were applied under steady laminar flow for 8 min. Biofilm response was evaluated by means of optical coherence tomography (OCT). Biofilm removal, biofilm disruption, rate of biofilm loss and disruption as well as bubble formation were assessed. One-way anova, Wilcoxon's signed-rank test and Kruskal–Wallis H test were performed for statistical analysis of the data. The level of significance was set at a ≤.05. Results: Increasing NaOCl concentration resulted in increased biofilm removal and disruption, higher rate of biofilm loss and disruption and increased bubble formation. Mixing HEDP with NaOCl caused a delay in the antibiofilm action of the latter, without compromising its antibiofilm efficacy. Conclusions: NaOCl concentration dictates the biofilm response irrespective of the presence of HEDP. The addition of HEDP resulted in a delay in the antibiofilm action of NaOCl. This delay affects the efficiency, but not the efficacy of the irrigant over time

Secreted products of oral bacteria and biofilms impede mineralization of apical papilla stem cells in TLR-, species-, and culture-dependent fashion

Regenerative endodontics exploits the mineralization potential of stem cells from the apical papilla (SCAPs) in order to promote root maturation of permanent immature teeth. SCAPs may encounter post-disinfection residual bacteria either in planktonic or in biofilm growth mode. Bacterial components bind to Toll-like receptors (TLRs) and trigger pro-inflammatory responses. We hypothesized that biofilm-triggered TLR activation affects the mineralization potential of human SCAPs. SCAPs were challenged with conditioned media derived from standardized dual-species biofilms and planktonic bacterial cultures and their inflammatory status and mineralization capacity were studied. Bacterial products from both growth modes (planktonic vs. biofilm) compromised cell viability, proliferation and mineralization capacity of SCAPs, but in a species- and growth mode-dependent fashion. While TLR4 expression remained unaffected, TLR2 expression was upregulated coinciding with a pro-inflammatory activation of SCAPs. Moreover, TLR and its downstream TGF-β-associated kinase (TAK1) appeared to be blocking mineralization, as inhibition of these factors restored it. In conclusion, bacterial products promoted the pro-inflammatory status and inhibited mineralization of human SCAPs in a TLR-, species-, and culture-dependent fashion. TLR2 emerged as the pivotal mediator of these responses and further research is warranted towards the judicious manipulation of SCAPs in order to modify the untoward events of TLR-priming and signaling

Cervical Level Biological Repair of the Access Opening after Regenerative Endodontic Procedures: Three Cases with the Same Repair Pattern

Introduction: This article describes the regenerative endodontic procedures applied in 3 cases of maxillary incisor necrosis that resulted in continuous root development, dentinal wall thickening, and cervical level biological repair of the access openings that was verified radiographically in 2 cases and clinically in 1 case. Methods: Three maxillary central incisors in 2 different patients were rendered necrotic after having dentin enamel fracture traumatic dental injuries. All teeth were treated with single- or multiple-visit regenerative endodontic procedures. Results: The 5- and 9-year follow-up evaluations revealed similar continuous root development, dentin wall thickening, and hard tissue biological repair of the wide access cavities. In the 9-year follow-up case, the calcium silicate cement was removed because of unacceptable discoloration. The hard tissue biological repair was visualized under the microscope and checked for its continuity with the axial walls, its resistance to displacement, and the presence of possible gaps. The repair tissue seemed to be yellowish in appearance with some brown niches of irregular texture, did not have detectable gaps, was firmly connected with the axial dentinal walls through a demarcated white line, and resisted ail displacement forces applied. The tooth was restored with bonded composite resin restoration after internal bleaching. The 10-year follow-up revealed satisfactory esthetics and uneventful soft and hard tissue healing. Conclusions: Cervical-level hard tissue repair of the access opening after the application of regenerative endodontic procedures in necrotic immature maxillary incisors might reinforce the weakened tooth structure to a great extent and warrants further investigation

Dental Stem Cells for Bone Regeneration

Successful bone repair is of immense clinical importance. Bone fractures are among the most common injuries encountered and inappropriate healing, estimated to 5–10 %, is expected to further increase along with the increase of the aging population. Cell-based therapies provide an alternative to bone grafting by preventing bone loss and reducing grafting morbidity. Apart from the bone marrow mesenchymal stem cells, a multitude of mesenchymal stem cells from different tissues exhibit osteogenic potential. Among them, mesenchymal stem cells of dental origin, initially isolated but not restricted to the dental pulp, seem to hold a prominent position in the field of bone regeneration. This chapter aims to present the different types of dental stem cells with emphasis on their osteogenic properties and in vivo applications in animal models of bone repair

Influence of specialty training and experience on endodontic decision making

INTRODUCTION: The purpose of this study was to compare decision-making choices among dentists with different levels of training. METHODS: Scanned periapical radiographs and a leaflet with relevant information of 17 endodontically treated teeth were mailed to 40 undergraduate students, 25 general practitioners, 20 postgraduate students, and 40 endodontists. All teeth were symptom-free. The hypothetical scenario referred to patients who sought treatment for first time and had noncontributory medical history. Five treatment options were given for each situation: (1) Extraction, (2) Surgical Retreatment, (3) Nonsurgical Retreatment, (4) Wait and See, and (5) No Therapy. Statistical analysis was performed by using multinomial logistic regression models. RESULTS: The overall response rate was 70.4%, with endodontists exhibiting the lowest response. The undergraduates gave 4 or 5 treatment solutions for all cases, in contrast to endodontists, who gave 2 or 3 treatment solutions in a percentage of 82%. Nonsurgical Retreatment predominated among the participants' choices. Significant differences were detected in (1) Extraction for postgraduate students (P = .008) and endodontists (P = .001), (2) Surgical Retreatment for general practitioners (P = .002), postgraduate students (P = .002), and endodontists (P = .001), and (3) Wait and See for postgraduate students (P = .023). CONCLUSIONS: Differences in specialty training and experience strongly influence endodontic decision making. Endodontists showed the most consistent agreement among the groups