Engineering the Future of Dental Health: Exploring Molecular Advancements in Dental Pulp Regeneration

Protected by the surrounding mineralized barriers of enamel, dentin, and cementum, dental pulp is a functionally versatile tissue that fulfills multiple roles. In addition to the perception of thermal and mechanical stimuli as a warning system and the deposition of dentin, the pulp performs a variety of immunological functions against invading microorganisms, both in terms of recognition and defense. Especially, in young patients, dental pulp is essential for the completion of root development, and early pulp necrosis results in fracture-prone teeth with fragile root walls [1–4]. Whether in young or adult patients, the loss of pulp tissue due to caries or trauma requires a therapeutic intervention by means of root canal treatment and obturation with a synthetic material. In recent years, innovative attempts have been made to regenerate dental pulp using advanced molecular biology techniques [5,6]. Promising approaches, based on tissue engineering and regenerative medicine, have been developed for this purpose [7,8]. In this context, stem cell-based or primarily cell-free approaches use specifically tailored scaffold materials and signaling molecules to achieve pulp regeneration, both in terms of tissue microarchitecture and functionality (Figure 1). Several of these approaches already take into account the requirements of potential clinical applications [9–11]

A Compilation of Study Models for Dental Pulp Regeneration

Efforts to heal damaged pulp tissue through tissue engineering have produced positive results in pilot trials. However, the differentiation between real regeneration and mere repair is not possible through clinical measures. Therefore, preclinical study models are still of great importance, both to gain insights into treatment outcomes on tissue and cell levels and to develop further concepts for dental pulp regeneration. This review aims at compiling information about different in vitro and in vivo ectopic, semiorthotopic, and orthotopic models. In this context, the differences between monolayer and three-dimensional cell cultures are discussed, a semiorthotopic transplantation model is introduced as an in vivo model for dental pulp regeneration, and finally, different animal models used for in vivo orthotopic investigations are presented

A critical analysis of clinical research methods to study regenerative endodontics

Regenerative endodontic treatment such as revitalization provides a treatment option for immature teeth with pulp necrosis. The main difference to the alternative procedure, the apical plug, is the induction of a blood clot inside the canal as a scaffold for healing and new tissue formation. Due to the biology-based and minimally-invasive nature of the treatment, revitalization has raised considerable interest in recent years. Whereas the procedure is fairly new and recommendations from endodontic societies have been in place only for a few years, the treatment protocol has evolved over the past two decades. Evidence has been created, not only from laboratory and animal work, but also from clinical studies including case reports, cohort studies and eventually prospective randomized controlled clinical trials, systematic reviews and meta-analyses. However, the research methods and clinical studies with subsequent reports oftentimes present with methodical limitations, which makes it difficult to objectively assess the value of this treatment modality. Several open questions remain, including the need for a more differentiated indication of revitalization after different traumatic injuries, the long-term prognosis of treated teeth and the true benefits for the patient. Therefore, this review aims to identify and reflect on such limitations, scrutinizing study design, diagnostic tools, procedural details and outcome parameters. A core outcome set is also proposed in this context, which can be considered in future clinical investigations. These considerations may lead to a more detailed and stringent planning and execution of future studies in order to create high-quality evidence for the treatment modality of revitalization and thus provide more robust data, create a larger body of knowledge for clinicians and further specify current recommendations

Three-Dimensional Human Cell Cultures for Cytotoxicity Testing of Dental Filling Materials

Cilj: Za uzgoj trodimenzionalnih kultura stanica radi testiranja citotoksičnosti stomatoloških materijala u testu dentinske barijere (DBT) dosad su se rabile “besmrtne” stanice goveđe pulpe. U ovom istraživanju ocijenili smo uporabu stanica dobivenih iz ljudske pulpe koje bi mogle preciznije simulirati kliničku situaciju. Također, testirali smo smolasti kompozitni materijal. Materijali i metode: SV40-transfektirane stanice ljudske pulpe (tHPC) uzgojene su u hidrogelu (fibrin, peptid, kolagen) te su određena mehanička svojstva i vitalnost stanica (MTT I WST-1). Na uzorcima stanica uzgojenih u kolagenu proveden je test proliferacije stanica nakon četiri tjedna (WST-1). Nakon 14 dana na uzgojenim trodimenzionalnim kulturama stanica u kolagenu proveden je DBT test s 200 μm debelim dentinskim diskovima. Nakon inkubacije od 24 sata pod perfuzijom (0.3ml/h) upotrijebljeni su materijali prema uputama proizvođača (1) President (Coltene): negativna (netoksična) kontrola, (2) CaGPG14 (ISO 7405): pozitivna (toksična) kontrola, (3) Tetric EvoCeram (Ivoclar Viadent) s Clearfil SEBond (Kuraray, referentni materijal), (4) N´Durance (Sepodont, testni materijal), (5) N´Durance s Clearfil SEBond. Ocijenjena je vitalnost stanica nakon inkubacije od 24 sata (WST-1). Izračunat je postotak relativne vitalnosti (negativna kontrola = 100%) i provedena je statistička analiza (Kruskal-Wallisov test, p<0.05). Rezultati: Hidrogelovi od fibrina i peptida pokazali su nedostatna mehanička svojstva za DBT. Kolagenski gel pokazao se pogodnim za trodimenzionalnu kulturu stanica tHCP do 21 dan. Nakon toga uzorci su preneseni na DBT analizu te su rezultati bili slični onima iz prijašnjih istraživanja s goveđim stanicama. DBT test, primjenjujući tHPC, u kolagenu nije pokazivao statistički značajne razlike između testiranih materijala s adhezivom i bez njega i referentnih smolastih kompozita. Zaključak: tHCP u kolagenu može nadomjestiti goveđe stanice u DBT testu. Testirani materijal ne uzrokuje oštećenje pulpe ako je prekrivena intaktnim slojem dentina.Objectives: So far, bovine immortalized pulp cells have been used as three dimensional cultures for cytotoxicity testing of filling materials in the dentin barrier test (DBT). In this study, the use of human pulp-derived cells was evaluated, which would better simulate the clinical situation, and a composite material with a new resin base was tested. Materials and Methods: SV40-transfected human pulp cells (tHPC) were cultured in hydrogels (fibrin, peptide, collagen) and mechanical properties and cell viability (MTT or WST-1) were determined. For cell cultures in collagen, a four week - proliferation assay was performed (WST-1). After 14 days of three-dimensional culture in collagen, tHPC were introduced into the DBT with 200 μm dentin disks. After a 24-hour incubation under perfusion (0.3 ml/h), the following materials were applied according to the manufacturers’ instructions (1) President (Coltene): negative (non-toxic) control, (2) CaGPG14 (ISO 7405): positive (toxic) control, (3) Tetric EvoCeram (Ivoclar Vivadent) with Clearfil SEBond (Kuraray, reference material), (4) N´Durance (Sepodont, test material), (5) N´Durance with Clearfil SEBond. Cell viability was determined after 24-hour incubation (WST-1). The percentage of relative viability was calculated (negative control=100%) and statistically analyzed (Kruskal-Wallis-test, p<0.05). Results: Fibrin and peptide gels had insufficient mechanical properties for the DBT. Collagen appeared suitable for three-dimensional cell culture of tHPC for up to 21 days. The cultures could be transferred to the DBT device and results for controls were similar to previous tests with bovine cells. The DBT using tHPC in the collagen showed no statistically significant difference between the test material with and without the adhesive and the reference resin composite. Conclusions: tHPC in collagen can replace bovine cells in the DBT. The tested filling material is not likely to cause pulp damage, if the pulp is covered by an intact dentin layer

Scaffolds for Dental Pulp Tissue Engineering

For tissue engineering strategies, the choice of an appropriate scaffold is the first and certainly a crucial step. A vast variety of biomaterials is available: natural or synthetic polymers, extracellular matrix, self-assembling systems, hydrogels, or bioceramics. Each material offers a unique chemistry, composition and structure, degradation profile, and possibility for modification. The role of the scaffold has changed from passive carrier toward a bioactive matrix, which can induce a desired cellular behavior. Tailor-made materials for specific applications can be created. Recent approaches to generate dental pulp rely on established materials, such as collagen, polyester, chitosan, or hydroxyapatite. Results after transplantation show soft connective tissue formation and newly generated dentin. For dentinpulp- complex engineering, aspects including vascularization, cell-matrix interactions, growth-factor incorporation, matrix degradation, mineralization, and contamination control should be considered. Self-assembling peptide hydrogels are an example of a smart material that can be modified to create customized matrices. Rational design of the peptide sequence allows for control of material stiffness, induction of mineral nucleation, or introduction of antibacterial activity. Cellular responses can be evoked by the incorporation of cell adhesion motifs, enzymecleavable sites, and suitable growth factors. The combination of inductive scaffold materials with stem cells might optimize the approaches for dentin-pulp complex regeneration

Inflammatory Response Mechanisms of the Dentine-Pulp Complex and the Periapical Tissues

The macroscopic and microscopic anatomy of the oral cavity is complex and unique in the human body. Soft-tissue structures are in close interaction with mineralized bone, but also dentine, cementum and enamel of our teeth. These are exposed to intense mechanical and chemical stress as well as to dense microbiologic colonization. Teeth are susceptible to damage, most commonly to caries, where microorganisms from the oral cavity degrade the mineralized tissues of enamel and dentine and invade the soft connective tissue at the core, the dental pulp. However, the pulp is well-equipped to sense and fend off bacteria and their products and mounts various and intricate defense mechanisms. The front rank is formed by a layer of odontoblasts, which line the pulp chamber towards the dentine. These highly specialized cells not only form mineralized tissue but exert important functions as barrier cells. They recognize pathogens early in the process, secrete antibacterial compounds and neutralize bacterial toxins, initiate the immune response and alert other key players of the host defense. As bacteria get closer to the pulp, additional cell types of the pulp, including fibroblasts, stem and immune cells, but also vascular and neuronal networks, contribute with a variety of distinct defense mechanisms, and inflammatory response mechanisms are critical for tissue homeostasis. Still, without therapeutic intervention, a deep carious lesion may lead to tissue necrosis, which allows bacteria to populate the root canal system and invade the periradicular bone via the apical foramen at the root tip. The periodontal tissues and alveolar bone react to the insult with an inflammatory response, most commonly by the formation of an apical granuloma. Healing can occur after pathogen removal, which is achieved by disinfection and obturation of the pulp space by root canal treatment. This review highlights the various mechanisms of pathogen recognition and defense of dental pulp cells and periradicular tissues, explains the different cell types involved in the immune response and discusses the mechanisms of healing and repair, pointing out the close links between inflammation and regeneration as well as between inflammation and potential malignant transformation

Pathophysiological mechanisms of root resorption after dental trauma: a systematic scoping review

Background The objective of this scoping review was to systematically explore the current knowledge of cellular and molecular processes that drive and control trauma-associated root resorption, to identify research gaps and to provide a basis for improved prevention and therapy. Methods Four major bibliographic databases were searched according to the research question up to February 2021 and supplemented manually. Reports on physiologic, histologic, anatomic and clinical aspects of root resorption following dental trauma were included. Duplicates were removed, the collected material was screened by title/abstract and assessed for eligibility based on the full text. Relevant aspects were extracted, organized and summarized. Results 846 papers were identified as relevant for a qualitative summary. Consideration of pathophysiological mechanisms concerning trauma-related root resorption in the literature is sparse. Whereas some forms of resorption have been explored thoroughly, the etiology of others, particularly invasive cervical resorption, is still under debate, resulting in inadequate diagnostics and heterogeneous clinical recommendations. Effective therapies for progressive replacement resorptions have not been established. Whereas the discovery of the RANKL/RANK/OPG system is essential to our understanding of resorptive processes, many questions regarding the functional regulation of osteo-/odontoclasts remain unanswered. Conclusions This scoping review provides an overview of existing evidence, but also identifies knowledge gaps that need to be addressed by continued laboratory and clinical research

An in vitro coculture approach to study the interplay between dental pulp cells and Streptococcus mutans

Aim To develop a new coculture system that allows exposure of dental pulp cells (DPCs) to Streptococcus mutans and dentine matrix proteins (eDMP) to study cellular interactions in dentine caries. Methodology Dental pulp cells and S. mutans were cocultured with or without eDMP for 72 h. Cell proliferation and viability were assessed by cell counting and MTT assays, while bacterial growth and viability were determined by CFU and LIVE/DEAD staining. Glucose catabolism and lactate excretion were measured photometrically as metabolic indicators. To evaluate the inflammatory response, the release of cytokines and growth factors (IL-6, IL-8, TGF-β1, VEGF) was determined by ELISA. Non-parametric statistical analyses were performed to compare all groups and time points (Mann–Whitney U test or Kruskal–Wallis test; α = .05). Results While eDMP and especially S. mutans reduced the number and viability of DPCs (p ≤ .0462), neither DPCs nor eDMP affected the growth and viability of S. mutans during coculture (p > .0546). The growth of S. mutans followed a common curve, but the death phase was not reached within 72 h. S. mutans consumed medium glucose in only 30 h, whereas in the absence of S. mutans, cells were able to catabolize glucose throughout 72 h, resulting in the corresponding amount of l-lactate. No change in medium pH was observed. S. mutans induced IL-6 production in DPCs (p ≤ .0011), whereas eDMP had no discernible effect (p > .7509). No significant changes in IL-8 were observed (p > .198). TGF-β1, available from eDMP supplementation, was reduced by DPCs over time. VEGF, on the other hand, was increased in all groups during coculture. Conclusions The results show that the coculture of DPCs and S. mutans is possible without functional impairment. The bacterially induced stimulation of proinflammatory and regenerative cytokines provides a basis for future investigations and the elucidation of molecular biological relationships in pulp defence against caries

In-vitro-cytotoxicity of self-adhesive dental restorative materials

Objectives Although the introduction of self-adhesive composites in restorative dentistry is very promising, the innovation of new materials also presents challenges and unknowns. Therefore, the aim of this study was to investigate the cytotoxicity of four different self-adhesive composites (SAC) in vitro and to compare them with resin-modified glass ionomer cements (RM-GIC), a more established group of materials. Methods Samples of the following materials were prepared according to ISO 7405/10993–12 and eluted in cell culture medium for 24 h at 37 °C: Vertise Flow, Fusio Liquid Dentin, Constic, Surefil One, Photac Fil and Fuji II LC. Primary human pulp cells were obtained from extracted wisdom teeth and cultured for 24 h with the extracts in serial dilutions. Cell viability was evaluated by MTT assay, membrane disruption was quantified by LDH assay and apoptosis was assessed by flow cytometry after annexin/PI staining. Results Two SAC (Constic and Vertise Flow) and one RM-GIC (Photac Fil) significantly reduced cell viability by more than 30% compared to the untreated control (p < 0.001). Disruptive cell morphological changes were observed and the cells showed signs of late apoptosis and necrosis in flow cytometry. Membrane disruption was not observed with any of the investigated materials. Conclusion Toxic effects occurred independently of the substance group and need to be considered in the development of materials with regard to clinical implications. Clinical Significance SAC have many beneficial qualities, however, the cytotoxic effects of certain products should be considered when applied in close proximity to the dental pulp, as is often required

TEGDMA Reduces Mineralization in Dental Pulp Cells

Direct application of dentin bonding agents onto the exposed pulp has been advocated, but in vivo studies indicate a lack of reparative dentin formation. Our objective was to investigate the role of triethylene glycol dimethacrylate (TEGDMA), a commonly used compound in dentin bonding agents, as a potential inhibitor of mineralization. Human pulp cells were exposed to different concentrations of TEGDMA, and expression of the mineralization-related genes collagen I, alkaline phosphatase, bone sialoprotein, osteocalcin, Runx2, and dentin sialophosphoprotein was analyzed. Gene expression studies by real-time polymerase chain-reaction revealed a concentration- and timedependent decrease of mineralization markers. A subtoxic TEGDMA concentration (0.3 mM) reduced expression levels by 5 to 20% after 4 hrs and by 50% after 12 hrs. Furthermore, alkaline phosphatase activity and calcium deposition were significantly lower in dental pulp cells treated with TEGDMA over 14 days. These findings indicate that even low TEGDMA concentrations might inhibit mineralization induced by dental pulp cells, thus impairing reparative dentin formation after pulp capping with dentin bonding agents