Research Models in Dentine Development and Regeneration.

Dentine is a major component of teeth and is responsible for many of their functions, such as mastication and neural sensation/transduction. Over the past decades, numerous studies have focused on dentine development and regeneration using a variety of research models, including in vivo, ex vivo and in vitro models. In vivo animal models play a crucial role in the exploration of biochemical factors that are involved in dentine development, whereas ex vivo and in vitro models contribute mainly to the identification of biophysical factors in dentine regeneration, of which mechanical force is most critical. In the present review, research models involved in studies related to dentine development and regeneration were screened from publications released in recent years and summarised comprehensively, particularly in vivo animal models including prokaryotic microinjection, Cre/LoxP, CRISPR/Cas9, ZFN and TALEN, and scaffold-based in vitro and ex vivo models. The latter were further divided by the interactive forces. Summarising these research models will not only benefit the development of future dentine-related studies but also provide hints regarding the evolution of novel dentine regeneration strategies.

Deciphering load attenuation mechanisms of the dentin-enamel junction: Insights from a viscoelastic constitutive model.

A considerable material discontinuity between the enamel and dentin might jeopardize the tooth's mechanical durability over time without the attenuation of the dentin-enamel junction (DEJ). However, the critical loading transmission mechanism at the DEJ remains understudied. This study aimed to define the extent and effective width of the DEJ, along with its mechanical competence. The presence of DEJ interphase layer was identified using a motif analysis based on the ion beam-transmission electron microscopy coupled with nanoindentation modulus mapping. For each region, nanoindentation load-displacement curves were recorded and mathematically analyzed using an appropriate viscoelastic constitutive model. The time-course of indenter penetration (creep) behavior of the tooth tissues can be mathematically approximated by the Kelvin-Voigt model in series, which determined the visco-contribution to the overall mechanical responses. Therefore, the elastic-plastic contribution can be distinguished from the overall mechanical responses of the tooth after subtracting the visco-contributions. During the loading period, the enamel behavior was dominated by elastic-plastic responses, while both the dentin and DEJ showed pronounced viscoelastic responses. The instantaneous modulus of the DEJ, which was measured by eliminating viscoelastic behavior from the raw load-displacement curve, was almost double that of the dentin. The DEJ was stiffer than the dentin, but it exhibited large viscoelastic motion even at the initial loading stage. This study revealed that the load attenuation competence of the DEJ, which involves extra energy expenditure, is mainly associated with its viscoelasticity. The mathematical analysis proposed here, performed on the nanoindentation creep behavior, could potentially augment the existing knowledge on hard-tissue biomechanics. STATEMENT OF SIGNIFICANCE: In this study, we undertake a rigorous mechanical characterization of the dentin-enamel junction (DEJ) using an advanced nanoindentation technique coupled with a pertinent viscoelastic constitutive model. Our approach unveils the substantial viscoelastic contribution of the DEJ during the initial indentation loading phase and offers an elaborate delineation of the DEJ interphase layer through sophisticated image analysis. These insights significantly augment our understanding of tooth durability. Importantly, our innovative mathematical analysis of creep behavior introduces a novel approach with profound implications for future research in the expansive field of hard-tissue biomechanics. The pioneering methodologies and findings presented in this work hold substantial potential to invigorate progress in biomaterials research and fuel further explorations into the functionality of biological tissues.

Functional expression of oxytocin receptors in pulp-dentin complex.

Dental pulp-derived stromal cells (DPSCs) are a crucial cell population for maintaining the tissue integrity of the pulp-dentin complex. The oxytocin receptor (OXTR), a member of the G protein-coupled receptor (GPCR) superfamily, plays versatile roles in diverse biological contexts. However, the role of OXTR in dental pulp has not yet been fully understood. Here, we demonstrate the biological functions and significance of OXTR in DPSCs through a multidisciplinary approach. Microarray data of 494 GPCR genes revealed high OXTR expression in human DPSCs (hDPSCs). Blocking OXTR activity increased the expression of osteogenic and odontogenic marker genes, promoting hDPSC differentiation. Additionally, we found that OXTR is involved in extracellular matrix (ECM) remodeling through the regulation of the gene expression related to ECM homeostasis. We further demonstrated that these genetic changes are mediated by trascriptional activity of Yes-associated protein (YAP). Based on the results, a preclinical experiment was performed using an animal model, demonstrating that the application of an OXTR inhibitor to damaged pulp induced significant hard tissue formation. These results provide new insight into the oxytocin-OXTR system in the regenerative process of pulp-dentin complex.

Scaffolds for Dentin-Pulp Complex Regeneration.

Background and Objectives: Regenerative dentistry aims to regenerate the pulp-dentin complex and restore those of its functions that have become compromised by pulp injury and/or inflammation. Scaffold-based techniques are a regeneration strategy that replicate a biological environment by utilizing a suitable scaffold, which is considered crucial for the successful regeneration of dental pulp. The aim of the present review is to address the main characteristics of the different scaffolds, as well as their application in dentin-pulp complex regeneration. Materials and Methods: A narrative review was conducted by two independent reviewers to answer the research question: What type of scaffolds can be used in dentin-pulp complex regeneration? An electronic search of PubMed, EMBASE and Cochrane library databases was undertaken. Keywords including "pulp-dentin regeneration scaffold" and "pulp-dentin complex regeneration" were used. To locate additional reports, reference mining of the identified papers was undertaken. Results: A wide variety of biomaterials is already available for tissue engineering and can be broadly categorized into two groups: (i) natural, and (ii) synthetic, scaffolds. Natural scaffolds often contain bioactive molecules, growth factors, and signaling cues that can positively influence cell behavior. These signaling molecules can promote specific cellular responses, such as cell proliferation and differentiation, crucial for effective tissue regeneration. Synthetic scaffolds offer flexibility in design and can be tailored to meet specific requirements, such as size, shape, and mechanical properties. Moreover, they can be functionalized with bioactive molecules, growth factors, or signaling cues to enhance their biological properties and the manufacturing process can be standardized, ensuring consistent quality for widespread clinical use. Conclusions: There is still a lack of evidence to determine the optimal scaffold composition that meets the specific requirements and complexities needed for effectively promoting dental pulp tissue engineering and achieving successful clinical outcomes.

Influencing factors of pulp-dentin complex regeneration and related biological strategies.

Regenerative endodontic therapy (RET) utilizing tissue engineering approach can promote the regeneration of pulp-dentin complex to restore pulp vascularization, neuralization, immune function and tubular dentin, therefore the regenerated pulp-dentin complex will have normal function. Multiple factors may significantly affect the efficacy of RET, including stem cells, biosignaling molecules and biomaterial scaffolds. Stem cells derived from dental tissues (such as dental pulp stem cells) exhibit certain advantages in RET. Combined application of multiple signaling molecules and activation of signal transduction pathways such as Wnt/ß-catenin and BMP/Smad play pivotal roles in enhancing the potential of stem cell migration, proliferation, odontoblastic differentiation, and nerve and blood vessel regeneration. Biomaterials suitable for RET include naturally-derived materials and artificially synthetic materials. Artificially synthetic materials should imitate natural tissues for biomimetic modification in order to realize the temporal and spatial regulation of pulp-dentin complex regeneration. The realization of pulp-dentin complex regeneration depends on two strategies: stem cell transplantation and stem cell homing. Stem cell homing strategy does not require the isolation and culture of stem cells in vitro, so is better for clinical application. However, in order to achieve the true regeneration of pulp-dentin complex, problems related to improving the success rate of stem cell homing and promoting their proliferation and differentiation need to be solved. This article reviews the influencing factors of pulp-dentin complex regeneration and related biological strategies, and discusses the future research direction of RET, to provide reference for clinical translation and application of RET.

Advances in scaffolds used for pulp-dentine complex tissue engineering: A narrative review.

BACKGROUND: Pulp-dentine complex regeneration via tissue engineering is a developing treatment modality that aims to replace necrotic pulps with newly formed healthy tissue inside the root canal. Designing and fabricating an appropriate scaffold is a crucial step in such a treatment. OBJECTIVES: The present study aimed to review recent advances in the design and fabrication of scaffolds for de novo regeneration of pulp-dentine complexes via tissue engineering approaches. METHODS: A literature search was conducted using PubMed, Europe PMC, Scopus and Google Scholar databases. To highlight bioengineering techniques for de novo regeneration of pulp-dentine complexes, both in vitro and in vivo studies were included, and clinical studies were excluded. RESULTS: In the present review, four main classes of scaffolds used to engineer pulp-dentine complexes, including bioceramic-based scaffolds, synthetic polymer-based scaffolds, natural polymer-based scaffolds and composite scaffolds, are covered. Additionally, recent advances in the design, fabrication and application of such scaffolds are analysed along with their advantages and limitations. Finally, the importance of vascular network establishment in the success of pulp-dentine complex regeneration and strategies used to create scaffolds to address this challenge are discussed. DISCUSSION: In the tissue engineering platform, scaffolds provide structural support for cells to adhere and proliferate and also regulate cell differentiation and metabolism. Up to now, considerable progress has been achieved in the field of pulp-dentine complex tissue engineering, and a spectrum of scaffolds ranging from bioceramic-based to naturally derived scaffolds has been fabricated. However, in designing a suitable scaffold for engineering pulp-dentine complexes, a variety of characteristic parameters related to biological, structural, physical and chemical features should be considered. CONCLUSION: The variety of biomaterials and fabrication techniques provides a great opportunity to address some of the requirements for scaffolds in regenerative endodontics. However, more studies are required to develop an ideal scaffold for use in a clinical setting.

BMP Signaling Pathway in Dentin Development and Diseases.

BMP signaling plays an important role in dentin development. BMPs and antagonists regulate odontoblast differentiation and downstream gene expression via canonical Smad and non-canonical Smad signaling pathways. The interaction of BMPs with their receptors leads to the formation of complexes and the transduction of signals to the canonical Smad signaling pathway (for example, BMP ligands, receptors, and Smads) and the non-canonical Smad signaling pathway (for example, MAPKs, p38, Erk, JNK, and PI3K/Akt) to regulate dental mesenchymal stem cell/progenitor proliferation and differentiation during dentin development and homeostasis. Both the canonical Smad and non-canonical Smad signaling pathways converge at transcription factors, such as Dlx3, Osx, Runx2, and others, to promote the differentiation of dental pulp mesenchymal cells into odontoblasts and downregulated gene expressions, such as those of DSPP and DMP1. Dysregulated BMP signaling causes a number of tooth disorders in humans. Mutation or knockout of BMP signaling-associated genes in mice results in dentin defects which enable a better understanding of the BMP signaling networks underlying odontoblast differentiation and dentin formation. This review summarizes the recent advances in our understanding of BMP signaling in odontoblast differentiation and dentin formation. It includes discussion of the expression of BMPs, their receptors, and the implicated downstream genes during dentinogenesis. In addition, the structures of BMPs, BMP receptors, antagonists, and dysregulation of BMP signaling pathways associated with dentin defects are described.

Dental Pulp Fibroblast: A Star Cell.

INTRODUCTION: Dental pulp fibroblasts (DPFs) are the most abundant cell type in the dental pulp. They play pivotal roles; however, they are often mistaken to be involved only in the repair and maintenance of this connective tissue. METHODS: We used the search terms "pulp fibroblast," "complement system proteins," "pulp inflammation," "angiogenesis," and "dentin pulp regeneration" to identify articles from the PubMed and Scopus databases. RESULTS: These sentinel cells produce all complement system proteins participating in defense processes, control of inflammation, and dentin-pulp regeneration; produce several proinflammatory cytokines and chemokines and express pattern-recognition receptors, demonstrating their involvement in immunoregulatory mechanisms; express neuropeptides and their receptors, playing an important role in neurogenic inflammation and dental pulp wound healing; secrete angiogenic growth factors as well as neurotrophic proteins, essential for dentin-pulp regeneration; regulate neuronal plasticity processes; and can sense the external environment. CONCLUSIONS: This review highlights that DPFs are more than mere passive cells in pulp biology and presents an integrative analysis of their roles and functions.

Utilización de dentina autóloga como material de regeneración. Rehabilitación de un paciente mediante tratamiento quirúrgico y prostodóncico con flujo digital / Utilization of autologous dentin as regeneration material. A patients’ rehabilitation by surgical and prosthodontic treatment with digital workflow

Introducción: Existen numerosos procedimientos para conseguir un lecho óseo adecuado para colocar implantes tras la pérdida de dientes naturales. En los últimos años se han propuesto técnicas para la preservación del lecho tras la extracción dental. Los injertos de dentina autóloga ofrecen un sustrato conveniente con propiedades osteoinductivas y osteogénicas óptimas para la regeneración alveolar. Objetivo: Se presenta un caso clínico de un paciente rehabilitado mediante un tratamiento quirúrgico y prostodóntico, y una actualización de la bibliografía en relación con los injertos de dentina autóloga. Caso clínico: Varón de 64 años sin antecedentes médicos ni hábitos patológicos, que presenta desgastes severos, inestabilidad oclusal y problemas estéticos. Se realiza una rehabilitación integral del paciente combinando una técnica quirúrgica de preservación alveolar con injerto de dentina autóloga, tras la cual se procede a la colocación de implantes, con un tratamiento protésico de coronas de zirconio, incrustaciones de disilicato de litio y reconstrucciones de composite. El tratamiento protésico se realiza en dos fases, pasando por una fase de provisionalización previa a la colocación de las restauraciones definitivas, empleando el flujo digital. A los 6 meses el paciente se encuentra satisfecho y con una función y estética óptima. Conclusiones: El injerto de dentina autóloga parece una alternativa eficaz y predecible como material de regeneración alveolar. Combinando esta técnica de preservación con una planificación digital, se puede maximizar el resultado del tratamiento rehabilitador, consiguiendo una mayor satisfacción del paciente. (AU)

Introduction: There are multiple procedures to achieve an adequate bone site for implant placement after teeth loss. In the last years, numerous techniques have been proposed for alveolar preservation. Dentin autologous grafts offer a convenient substrate with osteoinductive and osteogenic properties, which are optimum for alveolar regeneration. Objective: In this article, a clinical case of a patient rehabilitated by surgical and prosthodontic treatment, and a review of the literature regarding autologous dentin grafts is presented. Case report: 64 years old male, with no medical records or parafunctional habits, presents severe wear, occlusal instability and aesthetic problems. An integral rehabilitation is performed combining a surgical preservation technique with autologous dentin graft, after which the placement of the implants takes place, and a prosthodontic treatment with zirconium crowns, lithium disilicate inlays and composite restorations. The prosthodontic treatment is accomplished in two phases, going through a provisionalization phase previous to the placement of the definitive restorations, and digital workflow is used. 6 months later, the patient is satisfied, and function and aesthetic are optimum. Conclusions: Dentin autologous graft offers a predictable and effective alternative as a material for alveolar regeneration. Combining this preservation technique, with a good digital planification, results can be maximized and satisfaction for the patient can be increased. (AU)

Secreted Frizzled-Related Protein 1 Promotes Odontoblastic Differentiation and Reparative Dentin Formation in Dental Pulp Cells.

Direct pulp capping is an effective treatment for preserving dental pulp against carious or traumatic pulp exposure via the formation of protective reparative dentin by odontoblast-like cells. Reparative dentin formation can be stimulated by several signaling molecules; therefore, we investigated the effects of secreted frizzled-related protein (SFRP) 1 that was reported to be strongly expressed in odontoblasts of newborn molar tooth germs on odontoblastic differentiation and reparative dentin formation. In developing rat incisors, cells in the dental pulp, cervical loop, and inner enamel epithelium, as well as ameloblasts and preodontoblasts, weakly expressed Sfrp1; however, Sfrp1 was strongly expressed in mature odontoblasts. Human dental pulp cells (hDPCs) showed stronger expression of SFRP1 compared with periodontal ligament cells and gingival cells. SFRP1 knockdown in hDPCs abolished calcium chloride-induced mineralized nodule formation and odontoblast-related gene expression and decreased BMP-2 gene expression. Conversely, SFRP1 stimulation enhanced nodule formation and expression of BMP-2. Direct pulp capping treatment with SFRP1 induced the formation of a considerable amount of reparative dentin that has a structure similar to primary dentin. Our results indicate that SFRP1 is crucial for dentinogenesis and is important in promoting reparative dentin formation in response to injury.

Multifunctional Ca-Zn-Si-based micro-nano spheres with anti-infective, anti-inflammatory, and dentin regenerative properties for pulp capping application.

While pulp capping using a variety of materials has been applied clinically to preserve the health and vitality of the dental pulp and induce dentin repair no material meets all the anti-infection, anti-inflammation, and promoting pulp tissue regeneration criteria. Micro-nano materials of bioactive glasses (BG) with the biocompatibility and osteogenesis-promoting properties were developed for this study using Zn-doped bioactive glass (BGz) micro-nano spheres for dental pulp capping to control infection and inflammation and promote tissue regeneration. Of three key findings, the co-culture of Porphyromonas gingivalis showed that the BGz had an excellent antibacterial effect, and after being stimulated with BGz in vitro, macrophages showed a significant decrease of pro-inflammatory M1 markers compared with the undoped BG group. It is also noted that the conditioned medium derived from BGz-stimulated macrophages could significantly promote mineralized dentin formation of dental pulp cells (DPCs). In rats, acute pulp restoration experiments proved that BGz used as a pulp capping agent had excellent dentin regenerative properties. This work may provide a novel strategy to promote osteo/dentinogenic differentiation through regulating early inflammation, with potential applications in pulp capping.

The Role of GH/IGF Axis in Dento-Alveolar Complex from Development to Aging and Therapeutics: A Narrative Review.

The GH/IGF axis is a major regulator of bone formation and resorption and is essential to the achievement of normal skeleton growth and homeostasis. Beyond its key role in bone physiology, the GH/IGF axis has also major pleiotropic endocrine and autocrine/paracrine effects on mineralized tissues throughout life. This article aims to review the literature on GH, IGFs, IGF binding proteins, and their respective receptors in dental tissues, both epithelium (enamel) and mesenchyme (dentin, pulp, and tooth-supporting periodontium). The present review re-examines and refines the expression of the elements of the GH/IGF axis in oral tissues and their in vivo and in vitro mechanisms of action in different mineralizing cell types of the dento-alveolar complex including ameloblasts, odontoblasts, pulp cells, cementoblasts, periodontal ligament cells, and jaw osteoblasts focusing on cell-specific activities. Together, these data emphasize the determinant role of the GH/IGF axis in physiological and pathological development, morphometry, and aging of the teeth, the periodontium, and oral bones in humans, rodents, and other vertebrates. These advancements in oral biology have elicited an enormous interest among investigators to translate the fundamental discoveries on the GH/IGF axis into innovative strategies for targeted oral tissue therapies with local treatments, associated or not with materials, for orthodontics and the repair and regeneration of the dento-alveolar complex and oral bones.

Ontogenetic development of the holocephalan dentition: Morphological transitions of dentine in the absence of teeth.

Among the cartilaginous fishes (Chondrichthyes), the Holocephali are unique in that teeth are absent both in ontogeny and adult regenerative growth. Instead, the holocephalan dentition of ever-growing nonshedding dental plates is composed of dentine, trabecular in arrangement, forming spaces into which a novel hypermineralized dentine (whitlockin) is deposited. These tissue features form a variety of specific morphologies as the defining characters of dental plates in the three families of extant holocephalans. We demonstrate how this morphology changes through ontogenetic development with continuity between morphologies, through successive growth stages of the dentition represented by the dental plate. For example, rod-shaped whitlockin appears early, later transformed into the tritoral pad, including a regular arrangement of vascular canals and whitlockin forming with increasing mineralization (95%-98%). While the tritoral pads develop lingually, stacks of individual ovoids of whitlockin replace the rods in the more labial parts of the plate, again shaped by the forming trabecular dentine. The ability to make dentine into new, distinctive patterns is retained in the evolution of the Holocephali, despite the lack of teeth forming in development of the dentition. We propose that developmentally, odontogenic stem cells, retained through evolution, control the trabecular dentine formation within the dental plate, and transition to form whitlockin, throughout lifetime growth. Our model of cellular activity proposes a tight membrane of odontoblasts, having transformed to whitloblasts, that can control active influx of minerals to the rapidly mineralizing dentine, forming whitlockin. After the reduced whitloblast cells transition back to odontoblasts, they continue to monitor the levels of minerals (calcium, phosphate and magnesium) and at a slower rate of growth in the peritubate 'softer' dentine. This model explains the unique features of transitions within the holocephalan dental plate morphology.

Advanced in Vitro Experimental Models for Tissue Engineering-based Reconstruction of a 3D Dentin/pulp Complex: a Literature Review.

OBJECTIVE: Experimental procedures have been used to monitor cellular responses at the dentin/pulp interface. Aiming to divert from in vivo studies and oversimplified two-dimensional assays, three-dimensional (3D) models have been developed. This review provides an overview of existing literature, regarding 3D in vitro dentin/pulp reconstruction. MATERIAL & METHODS: PubMed, Scopus, Cochrane Library and Web of Science- were systematically searched for attributes between 1998 and 2020. The search focused on articles on the development of three-dimensional tools for the reconstruction of a dentin/pulp complex under in vitro conditions, which were then screened and qualitatively assessed. Article grouping according to mode of implementation, resulted in five categories: the customised cell perfusion chamber (CPC) (n = 8), the tooth bud model (TBM) (n = 3), the 3D dentin/pulp complex manufactured by tissue engineering (DPC) (n = 6), the entire tooth culture (ETC) (n = 4) and the tooth slice culture model (TSC) (n = 5). RESULTS: A total of 26 publications, applying nine and eight substances for pulp and dentin representation respectively, were included. Natural materials and dentin components were the most widely utilized. The most diverse category was the DPC, while the CPC group was the test with the highest longevity. The most consistent categories were the ETC and TSC models, while the TBM presented as the most complete de novo approach. CONCLUSIONS: All studies presented with experimental protocols with potential upgrades. Solving the limitations of each category will provide a complete in vitro testing and monitoring tool of dental responses to exogenous inputs. CLINICAL RELEVANCE: The 3D dentin/pulp complexes are valid supplementary tools for in vivo studies and clinical testing. Graphical Abstract.

Biomechanical behavior of endocrowns vs fiber post-core-crown vs cast post-core-crown for the restoration of maxillary central incisors with 1 mm and 2 mm ferrule height: A 3D static linear finite element analysis.

To analyze the stress distribution of the maxillary central incisor with oblique fracture, repaired by different methods, using 3-dimensional finite element analysis. From the biomechanical point of view, it is expected to provide a reference for clinical selection of restoration method which is more conducive to stress distribution and preservation of dental tissue as much as possible.Use cone beam CT and finite element software to establish the finite element models of the maxillary central incisor with oblique fracture, and then create models according to 5 repairing methods(A. fiber post-core-crown group; B. cast post-core-crown group; C.3 mm deep endocrown; D.4 mm deep endocrown; E.5 mm deep endocrown)after root canal treatment, and analyze the Von Mises equivalent stress and maximum principal stress distribution and peak value of each model.When the height of dentin ferrule was fixed, the value of the Von Mises equivalent stress and the maximum principal stress in residual tooth tissue: group A was the highest, and there was no significant difference in group B, C, D and E. And the stress distribution area of 5 groups were the same. In prosthodontic layer: group B was the highest, while group A was the lowest, and the stress peak slightly increased with the increase of depth in group C, D and E. And the 5 groups were with the same stress distribution area as well. In adhesive layer: group A was the highest, while group B was the lowest, and there was little difference among group C, D and E. Group A was concentrated in 1/3 of the post tip, while group B,C,D and E were concentrated in 1/3 of the post and the post tips.Complete and high enough dentin ferrule is a requirement for repairing heavily defected maxillary central incisor with fiber post-core crown and cast post-core crown. When the dentin ferrule is incomplete, the stress distribution of the endocrown is more excellent than post-core-crown. And the endocrown with a depth of 3 mm retainer may be the best repair method. As for post-core crown restoration, the cast post-core crown is more favorable for the uniform distribution of residual tooth tissue than the fiber post-core crown.

Evaluation of the regenerative potential of dentin conditioning and naturally derived scaffold for necrotic immature permanent teeth in a dog model.

This study evaluated the outcome of partial exposure of dentin matrix to ethylenediaminetetraacetic acid (EDTA) and application of platelet-rich fibrin (PRF) scaffold on regeneration of necrotic immature permanent teeth in a dog model. The present study was carried out on 216 permanent immature roots in nine mongrel dogs aged 6-9 months. Pulp necrosis and periapical pathosis were induced in 180 roots. These roots were divided into five equal groups (36 roots each) according to the treatment protocol: group I: blood clot; group II: 17% EDTA solution and blood clot; group III: PRF; group IV: 17% EDTA solution and PRF; and group V: without treatment (positive control). The negative control group (group VI) represented 36 untouched normal roots for normal maturation. The groups were followed up for 1, 2 and 3 months (subgroups). Maturation of the roots was monitored by radiography and histopathology. All data were statistically analysed. Group IV exhibited the highest increase in root length and thickness, decrease in apical diameter, the highest score of vital tissue infiltration and least inflammatory scores. There was a significant difference regarding the increase in root length and thickness and decrease in apical diameter in all subgroups of the experimental and negative control groups (P ≤ .05). PRF has a better regenerative potential than the blood clot during treatment of immature permanent teeth with necrotic pulp. Inclusion of 17% EDTA solution as a final irrigation enhances the regenerative potential of both PRF and blood clot.

RGD- and VEGF-Mimetic Peptide Epitope-Functionalized Self-Assembling Peptide Hydrogels Promote Dentin-Pulp Complex Regeneration.

INTRODUCTION: Cell-based tissue engineering is a promising method for dentin-pulp complex (DPC) regeneration. The challenges associated with DPC regeneration include the generation of a suitable microenvironment that facilitates the complete odontogenic differentiation of dental pulp stem cells (DPSCs) and the rapid induction of angiogenesis. Thus, the survival and subsequent differentiation of DPSCs are limited. Extracellular matrix (ECM)-like biomimetic hydrogels composed of self-assembling peptides (SAPs) were developed to provide an appropriate microenvironment for DPSCs. For functional DPC regeneration, the most important considerations are to provide an environment that promotes the adequate attachment of DPSCs and rapid vascularization of the regenerating pulp. Morphogenic signals in the form of growth factors (GFs) have been incorporated into SAPs to promote productive DPSC behaviors. However, the use of GFs has several drawbacks. We envision using a scaffold with SAPs coupled with long-term factors to increase DPSC attachment and vascularization as a method to address this challenge. METHODS: In this study, we developed synthetic material for an SAP-based scaffold with RGD- and vascular endothelial growth factor (VEGF)-mimetic peptide epitopes with the dual functions of dentin and pulp regeneration. DPSCs and human umbilical vein endothelial cells (HUVECs) were used to evaluate the biological effects of SAP-based scaffolds. Furthermore, the pulpotomized molar rat model was employed to test the reparative and regenerative effects of SAP-based scaffolds. RESULTS: This scaffold simultaneously presented RGD- and VEGF-mimetic peptide epitopes and provided a 3D microenvironment for DPSCs. DPSCs grown on this composite scaffold exhibited significantly improved survival and angiogenic and odontogenic differentiation in the multifunctionalized group in vitro. Histological and functional evaluations of a partially pulpotomized rat model revealed that the multifunctionalized scaffold was superior to other options with respect to stimulating pulp recovery and dentin regeneration in vivo. CONCLUSION: Based on our data obtained with the functionalized SAP scaffold, a 3D microenvironment that supports stem cell adhesion and angiogenesis was generated that has great potential for dental pulp tissue engineering and regeneration.

A structural biorhythm related to human sexual dimorphism.

Life on earth is regulated by biological rhythms, some of which oscillate with a circadian, monthly or lunar cycle. Recent research suggests that there is a near weekly biorhythm that may exert an influence on human skeletal growth. Evidence for the timing of this biorhythm is retained in tooth enamel as the periodicity of Retzius lines. Studies report that Retzius periodicity (RP) relates to adult human stature and enamel thickness. Adult human stature is sexually dimorphic, and so is enamel thickness of maxillary third molars (M3) but not mandibular M3. Yet, previous studies report sex differences in RP are apparent in some populations but not others, and it is unknown if dimorphism in enamel thickness relates to RP. To further our understanding of this biorhythm we analysed sex-related variation in RP and its relationship with enamel thickness in a sample of M3's (n = 94) from adults in Northern Britain. Results reveal RP was significantly higher in our sample of female molars compared to those of males, which is consistent with the previously reported correlation between the biorhythm and adult stature. The RP of maxillary M3 related to sex differences in enamel thickness, but this relationship was not present in mandibular M3. Our results support previous findings suggesting that this biorhythm is sexually dimorphic and provide the first evidence that RP may be one factor influencing sex differences in enamel thickness. Our study also shows that correlations between RP and enamel thickness appear to be most readily detected for tooth types with sufficiently wide ranges of enamel thickness variation, as is the case for maxillary but not mandibular M3. Achieving a sufficient sample size was critical for detecting a sex difference in periodicity.

Exosome-like vesicles derived from Hertwig's epithelial root sheath cells promote the regeneration of dentin-pulp tissue.

Background: The formation of dentin-pulp involves complex epithelial-mesenchymal interactions between Hertwig's epithelial root sheath cells (HERS) and dental papilla cells (DPCs). Earlier studies have identified some of the regulatory molecules participating in the crosstalk between HERS and DPCs and the formation of dentin-pulp. In the present study we focused on the role of HERS-secreted exosomes in DPCs and the formation of dentin-pulp. Specifically, we hypothesized that exosome-like vesicles (ELVs) might mediate the function of HERS and trigger lineage-specific differentiation of dental mesenchymal cells. To test our hypothesis, we evaluated the potential of ELVs derived from a HERS cell line (ELVs-H1) in inducing in vitro and in vivo differentiation of DPCs. Methods: ELVs-H1 were characterized using transmission electron microscopy and dynamic light scattering. The proliferation, migration, and odontoblast differentiation of DPCs after treatment with ELVs-H1, was detected by CCK8, transwell, ALP, and mineralization assays, respectively. Real time PCR and western blotting were used to detect gene and protein expression. For in vivo studies, DPC cells were mixed with collagen gel combined with or without ELVs and transplanted into the renal capsule of rats or subcutaneously into nude mice. HE staining and immunostaining were used to verify the regeneration of dentin-pulp and expression of odontoblast differentiation markers. Results: ELVs-H1 promoted the migration and proliferation of DPCs and also induced odontogenic differentiation and activation of Wnt/ß-catenin signaling. ELVs-H1 also contributed to tube formation and neural differentiation in vitro. In addition, ELVs-H1 attached to the collagen gel, and were slowly released and endocytosed by DPCs, enhancing cell survival. ELVs-H1 together with DPCs triggered regeneration of dental pulp-dentin like tissue comprised of hard (reparative dentin-like tissue) and soft (blood vessels and neurons) tissue, in an in vivo tooth root slice model. Conclusion: Our data highlighted the potential of ELVs-H1 as biomimetic tools in providing a microenvironment for specific differentiation of dental mesenchymal stem cells. From a developmental perspective, these vesicles might be considered as novel mediators facilitating the epithelial-mesenchymal crosstalk. Their instructive potency might be exploited for the regeneration of dental pulp-dentin tissues.

What do rates of deposition of dental cementum tell us? Functional and evolutionary hypotheses in red deer.

Cementum is a bone connective tissue that provides a flexible attachment for the tooth to the alveolar bone in many mammalian species. It does not undergo continuous remodelling, unlike non-dental bone, which combined with its growth pattern of seasonal layering makes this tissue uniquely suitable as a proxy for tracking changes in body repair investment throughout an animal´s life. We tested functional and sexual selection hypotheses on the rate of cementum deposition related to the highly polygynous mating strategy of red deer. We used a sample of 156 first lower molars from wild Scottish red deer of known age between 1 and 17 years old, approximately balanced by sex and age class. Cementum deposition on the inter-radicular pad increased with age at a constant average rate of 0.26 mm per year, with no significant differences between sexes. Cementum deposition was independent of (i) tooth wear, other than that associated with age, and (ii) enamel and dentine micro-hardness. The results partially supported the hypothesis that the main function of cementum is the repositioning of the tooth to maintain opposing teeth in occlusion. However, teeth that had more wear or males´ teeth that had faster rates of tooth wear than those of females did not present the expected higher rates of cementum deposition.