Effects of dodecacalcium hepta-aluminate content on the setting time, compressive strength, alkalinity, and cytocompatibility of tricalcium silicate cement

Abstract Objective This study aimed to investigate the effects of dodecacalcium hepta-aluminate (C12A7) content on some physicochemical properties and cytocompatibility of tricalcium silicate (C3S) cement using human dental pulp cells (hDPCs). Material and Methods High purity C3S cement was manufactured by a solid phase method. C12A7 was mixed with the cement in proportions of 0, 5, 8, and 10 wt% (C12A7-0, −5, −8, and −10, respectively). Physicochemical properties including initial setting time, compressive strength, and alkalinity were evaluated. Cytocompatibility was assessed with cell viability tests and cell number counts. Statistical analysis was performed by using one-way analysis of variance (ANOVA) and Tukey's test (p<0.05). Results The initial setting time of C3S-based cement was shorter in the presence of C12A7 (p<0.05). After 1 day, C12A7-5 showed significantly higher compressive strength than the other groups (p<0.05). After 7 days, the compressive strength of C12A7-5 was similar to that of C12A7-0, whereas other groups showed strength lower than C12A7-0. The pH values of all tested groups showed no significant differences after 1 day (p>0.05). The C12A7-5 group showed similar cell viability to the C12A7-0 group (p>0.05), while the other experimental groups showed lower values compared to C12A7-0 group (p<0.05). The number of cells grown on the C12A7-5 specimen was higher than that on C12A7-8 and −10 (p<0.05). Conclusions The addition of C12A7 to C3S cement at a proportion of 5% resulted in rapid initial setting time and higher compressive strength with no adverse effects on cytocompatibility.

Decellularized Human Dental Pulp as a Scaffold for Regenerative Endodontics.

Teeth undergo postnatal organogenesis relatively late in life and only complete full maturation a few years after the crown first erupts in the oral cavity. At this stage, development can be arrested if the tooth organ is damaged by either trauma or caries. Regenerative endodontic procedures (REPs) are a treatment alternative to conventional root canal treatment for immature teeth. These procedures rely on the transfer of apically positioned stem cells, including stem cells of the apical papilla (SCAP), into the root canal system. Although clinical success has been reported for these procedures, the predictability of expected outcomes and the organization of the newly formed tissues are affected by the lack of an available suitable scaffold that mimics the complexity of the dental pulp extracellular matrix (ECM). In this study, we evaluated 3 methods of decellularization of human dental pulp to be used as a potential autograft scaffold. Tooth slices of human healthy extracted third molars were decellularized by 3 different methods. One of the methods generated the maximum observed decellularization with minimal impact on the ECM composition and organization. Furthermore, recellularization of the scaffold supported the proliferation of SCAP throughout the scaffold with differentiation into odontoblast-like cells near the dentinal walls. Thus, this study reports that human dental pulp from healthy extracted teeth can be successfully decellularized, and the resulting scaffold supports the proliferation and differentiation of SCAP. The future application of this form of an autograft in REPs can fulfill a yet unmet need for a suitable scaffold, potentially improving clinical outcomes and ultimately promoting the survival and function of teeth with otherwise poor prognosis.

Assessment of Pulp Regeneration Induced by Stem Cell Therapy by Magnetic Resonance Imaging.

INTRODUCTION: This study was designed to evaluate the usefulness of magnetic resonance imaging (MRI) to assess the regeneration of pulp tissue. METHODS: Mobilized dental pulp stem cells and granulocyte colony-stimulating factor with collagen were transplanted into mature pulpectomized teeth for pulp regeneration (n = 4). The controls consisted of pulpectomized teeth with or without collagen and normal teeth with intact pulp tissue (n = 4, each). The signal intensity (SI) of MRI using T2 sequences was compared after the extraction of teeth in dogs. MRI was correlated with the corresponding histologic findings. RESULTS: Pulp tissue was fully regenerated 90 days after cell transplantation. On the other hand, the root canal was empty in the control collagen-transplanted teeth at 90 days. The SI of the normal teeth was significantly higher than that of nonvital pulpectomized teeth and the controls of collagen transplanted teeth at 90 days. The stem cell transplanted teeth showed a gradual decrease in the SI until 180 days at which time the SI was similar to that in the normal teeth and significantly higher than that in the teeth transplanted with collagen alone without the stem cells. CONCLUSIONS: The changes in the SI of the pulplike tissue were consistent with the histologic findings, showing the potential usefulness of the noninvasive method to serially access the efficacy of pulp regenerative therapy.

Enterococcus faecalis Inhibits Osteoblast Differentiation and Induces Chemokine Expression.

INTRODUCTION: Enterococcus faecalis is commonly found in root canals of patients with refractory apical periodontitis, often accompanying inflammation and malfunctioning bone regeneration. In this study, we investigated the effect of E. faecalis on osteoblast differentiation and the ability to induce chemokine expression to recruit inflammatory cells. METHODS: Osteoblast precursors from mouse calvaria were differentiated into osteoblasts with ascorbic acid and ß-glycerophosphate in the absence or presence of heat-killed E. faecalis (HKEF). Alizarin red S staining was performed to determine the degree of mineralization. Reporter gene and reverse-transcription polymerase chain reaction assays were performed to examine the activity of the Runx2 transcription factor and the expression of osteogenic marker genes, respectively. Secretion of the chemokines keratinocyte-derived chemokine and monocyte chemotactic protein-1 was measured by the enzyme-linked immunosorbent assay, and their functions were analyzed by measuring the migration of peripheral blood mononuclear cells using a transwell system. RESULTS: HKEF inhibited osteoblast mineralization and Runx2 transcriptional activity, which are typical features of osteoblast differentiation. HKEF also decreased the expression of Runx2, osterix, ß-catenin, osteocalcin, and type I collagen. Interestingly, however, the expression of keratinocyte-derived chemokine and monocyte chemotactic protein-1 was increased by HKEF, and the culture supernatant of HKEF-stimulated osteoblasts increased the transmigration of peripheral blood mononuclear cells. CONCLUSIONS: HKEF inhibits osteoblast differentiation and induces chemokine expression, which might be involved in refractory apical periodontitis and bone loss.

Mesenchymal dental pulp cells attenuate dentin resorption in homeostasis.

Dentin in permanent teeth rarely undergoes resorption in development, homeostasis, or aging, in contrast to bone that undergoes periodic resorption/remodeling. The authors hypothesized that cells in the mesenchymal compartment of dental pulp attenuate osteoclastogenesis. Mononucleated and adherent cells from donor-matched rat dental pulp (dental pulp cells [DPCs]) and alveolar bone (alveolar bone cells [ABCs]) were isolated and separately cocultured with primary rat splenocytes. Primary splenocytes readily aggregated and formed osteoclast-like cells in chemically defined osteoclastogenesis medium with 20 ng/mL of macrophage colony-stimulating factor (M-CSF) and 50 ng/mL of receptor activator of nuclear factor κB ligand (RANKL). Strikingly, DPCs attenuated osteoclastogenesis when cocultured with primary splenocytes, whereas ABCs slightly but significantly promoted osteoclastogenesis. DPCs yielded ~20-fold lower RANKL expression but >2-fold higher osteoprotegerin (OPG) expression than donor-matched ABCs, yielding a RANKL/OPG ratio of 41:1 (ABCs:DPCs). Vitamin D3 significantly promoted RANKL expression in ABCs and OPG in DPCs. In vivo, rat maxillary incisors were atraumatically extracted (without any tooth fractures), followed by retrograde pulpectomy to remove DPCs and immediate replantation into the extraction sockets to allow repopulation of the surgically treated root canal with periodontal and alveolar bone-derived cells. After 8 wk, multiple dentin/root resorption lacunae were present in root dentin with robust RANKL and OPG expression. There were areas of dentin resoprtion alternating with areas of osteodentin formation in root dentin surface in the observed 8 wk. These findings suggest that DPCs of the mesenchymal compartment have an innate ability to attenuate osteoclastogenesis and that this innate ability may be responsible for the absence of dentin resorption in homeostasis. Mesenchymal attenuation of dentin resorption may have implications in internal resorption in the root canal, pulp/dentin regeneration, and root resorption in orthodontic tooth movement.

Scaffold-free Prevascularized Microtissue Spheroids for Pulp Regeneration.

Creating an optimal microenvironment that mimics the extracellular matrix (ECM) of natural pulp and securing an adequate blood supply for the survival of cell transplants are major hurdles that need to be overcome in dental pulp regeneration. However, many currently available scaffolds fail to mimic essential functions of natural ECM. The present study investigated a novel approach involving the use of scaffold-free microtissue spheroids of dental pulp stem cells (DPSCs) prevascularized by human umbilical vein endothelial cells (HUVECs) in pulp regeneration. In vitro-fabricated microtissue spheroids were inserted into the canal space of tooth-root slices and were implanted subcutaneously into immunodeficient mice. Histological examination revealed that, after four-week implantation, tooth-root slices containing microtissue spheroids resulted in well-vascularized and cellular pulp-like tissues, compared with empty tooth-root slices, which were filled with only subcutaneous fat tissue. Immunohistochemical staining indicated that the tissue found in the tooth-root slices was of human origin, as characterized by the expression of human mitochondria, and contained odontoblast-like cells organized along the dentin, as assessed by immunostaining for nestin and dentin sialoprotein (DSP). Vascular structures formed by HUVECs in vitro were successfully anastomosed with the host vasculature upon transplantation in vivo, as shown by immunostaining for human CD31. Collectively, these findings demonstrate that prevascularized, scaffold-free, microtissue spheroids can successfully regenerate vascular dental pulp-like tissue and also highlight the significance of the microtissue microenvironment as an optimal environment for successful pulp-regeneration strategies.

Pulp stem cells: implication in reparative dentin formation.

Many dental pulp stem cells are neural crest derivatives essential for lifelong maintenance of tooth functions and homeostasis as well as tooth repair. These cells may be directly implicated in the healing process or indirectly involved in cell-to-cell diffusion of paracrine messages to resident (pulpoblasts) or nonresident cells (migrating mesenchymal cells). The identity of the pulp progenitors and the mechanisms sustaining their regenerative capacity remain largely unknown. Taking advantage of the A4 cell line, a multipotent stem cell derived from the molar pulp of mouse embryo, we investigated the capacity of these pulp-derived precursors to induce in vivo the formation of a reparative dentin-like structure upon implantation within the pulp of a rodent incisor or a first maxillary molar after surgical exposure. One month after the pulp injury alone, a nonmineralized fibrous matrix filled the mesial part of the coronal pulp chamber. Upon A4 cell implantation, a mineralized osteodentin was formed in the implantation site without affecting the structure and vitality of the residual pulp in the central and distal parts of the pulp chamber. These results show that dental pulp stem cells can induce the formation of reparative dentin and therefore constitute a useful tool for pulp therapies. Finally, reparative dentin was also built up when A4 progenitors were performed by alginate beads, suggesting that alginate is a suitable carrier for cell implantation in teeth.

A novel combinatorial therapy with pulp stem cells and granulocyte colony-stimulating factor for total pulp regeneration.

Treatment of deep caries with pulpitis is a major challenge in dentistry. Stem cell therapy represents a potential strategy to regenerate the dentin-pulp complex, enabling conservation and restoration of teeth. The objective of this study was to assess the efficacy and safety of pulp stem cell transplantation as a prelude for the impending clinical trials. Clinical-grade pulp stem cells were isolated and expanded according to good manufacturing practice conditions. The absence of contamination, abnormalities/aberrations in karyotype, and tumor formation after transplantation in an immunodeficient mouse ensured excellent quality control. After autologous transplantation of pulp stem cells with granulocyte-colony stimulating factor (G-CSF) in a dog pulpectomized tooth, regenerated pulp tissue including vasculature and innervation completely filled in the root canal, and regenerated dentin was formed in the coronal part and prevented microleakage up to day 180. Transplantation of pulp stem cells with G-CSF yielded a significantly larger amount of regenerated dentin-pulp complex compared with transplantation of G-CSF or stem cells alone. Also noteworthy was the reduction in the number of inflammatory cells and apoptotic cells and the significant increase in neurite outgrowth compared with results without G-CSF. The transplanted stem cells expressed angiogenic/neurotrophic factors. It is significant that G-CSF together with conditioned medium of pulp stem cells stimulated cell migration and neurite outgrowth, prevented cell death, and promoted immunosuppression in vitro. Furthermore, there was no evidence of toxicity or adverse events. In conclusion, the combinatorial trophic effects of pulp stem cells and G-CSF are of immediate utility for pulp/dentin regeneration, demonstrating the prerequisites of safety and efficacy critical for clinical applications.

Pulpal progenitors and dentin repair.

Mesenchymal stem cells are present in the dental pulp. They have been shown to contribute to dentin-like tissue formation in vitro and to participate in bone repair after a mandibular lesion. However, their capacity to contribute efficiently to reparative dentin formation after pulp lesion has never been explored. After pulp exposure, we have identified proliferative cells within 3 zones. In the crown, zone I is near the cavity, and zone II corresponds to the isthmus between the mesial and central pulp. In the root, zone III, near the apex, at a distance from the inflammatory site, contains mitotic stromal cells which may represent a source of progenitor cells. Stem-cell-based strategies are promising treatments for tissue injury in dentistry. Our experiments focused on (1) location of stem cells induced to leave their quiescent state early after pulp injury and (2) implantation of pulp progenitors, a substitute for classic endodontic treatments, paving the way for pulp stem-cell-based therapies.

Complete pulp regeneration after pulpectomy by transplantation of CD105+ stem cells with stromal cell-derived factor-1.

Loss of pulp due to caries and pulpitis leads to loss of teeth and reduced quality of life. Thus, there is an unmet need for regeneration of pulp. A promising approach is stem cell therapy. Autologous pulp stem/progenitor (CD105(+)) cells were transplanted into a root canal with stromal cell-derived factor-1 (SDF-1) after pulpectomy in mature teeth with complete apical closure in dogs. The root canal was successfully filled with regenerated pulp including nerves and vasculature by day 14, followed by new dentin formation along the dentinal wall. The newly regenerated tissue was significantly larger in the transplantation of pulp CD105(+) cells with SDF-1 compared with those of adipose CD105(+) cells with SDF-1 or unfractionated total pulp cells with SDF-1. The pulp CD105(+) cells highly expressed angiogenic/neurotrophic factors compared with other cells and localized in the vicinity of newly formed capillaries after transplantation, demonstrating its potent trophic effects on neovascularization. Two-dimensional electrophoretic analyses and real-time reverse transcription-polymerase chain reaction analyses demonstrated that the qualitative and quantitative protein and mRNA expression patterns of the regenerated pulp were similar to those of normal pulp. Thus, this novel stem cell therapy is the first demonstration of complete pulp regeneration, implying novel treatment to preserve and save teeth.

Evaluation of the delivery of mesenchymal stem cells into the root canal space of necrotic immature teeth after clinical regenerative endodontic procedure.

INTRODUCTION: Immature teeth with open apices treated with conventional nonsurgical root canal treatment often have a poor prognosis as a result of the increased risk of fracture and susceptibility to recontamination. Regenerative endodontics represents a new treatment modality that focuses on reestablishment of pulp vitality and continued root development. This clinical procedure relies on the intracanal delivery of a blood clot (scaffold), growth factors (possibly from platelets and dentin), and stem cells. However, to date, the clinical presence of stem cells in the canal space after this procedure has not been demonstrated. The purpose of this clinical study was to evaluate whether regenerative endodontic procedures are able to deliver stem cells into the canal space of immature teeth in young patients and to identify the possible tissue origin for these cells. METHODS: After informed consent, the first appointment consisted of NaOCl irrigation and treatment with a triple antibiotic paste. One month later, the root canal space was irrigated with sterile saline, and bleeding was evoked with collection of samples on paper points. Real-time reverse-transcription polymerase chain reaction and immunocytochemistry were conducted to compare the gene transcripts and proteins found in the root canal sample with levels found in the systemic circulation. RESULTS: Molecular analyses of blood collected from the canal system indicated the significant accumulation of transcripts for the stem cell markers CD73 and CD105 (up to 600-fold), compared with levels found in the systemic blood. Furthermore, this effect was selective because there was no change in expression of the differentiation markers ALK-P, DSPP, ZBTB16, and CD14. Histologic analyses demonstrated that the delivered cells expressed both CD105 and STRO-1, markers for a subpopulation of mesenchymal stem cells. CONCLUSIONS: Collectively, these findings demonstrate that the evoked-bleeding step in regenerative procedures triggers the significant accumulation of undifferentiated stem cells into the canal space where these cells might contribute to the regeneration of pulpal tissues seen after antibiotic paste therapy of the immature tooth with pulpal necrosis.

Stem/progenitor cell-mediated de novo regeneration of dental pulp with newly deposited continuous layer of dentin in an in vivo model.

The ultimate goal of this study is to regenerate lost dental pulp and dentin via stem/progenitor cell-based approaches and tissue engineering technologies. In this study, we tested the possibility of regenerating vascularized human dental pulp in emptied root canal space and producing new dentin on existing dentinal walls using a stem/progenitor cell-mediated approach with a human root fragment and an immunocompromised mouse model. Stem/progenitor cells from apical papilla and dental pulp stem cells were isolated, characterized, seeded onto synthetic scaffolds consisting of poly-D,L-lactide/glycolide, inserted into the tooth fragments, and transplanted into mice. Our results showed that the root canal space was filled entirely by a pulp-like tissue with well-established vascularity. In addition, a continuous layer of dentin-like tissue was deposited onto the canal dentinal wall. This dentin-like structure appeared to be produced by a layer of newly formed odontoblast-like cells expressing dentin sialophosphoprotein, bone sialoprotein, alkaline phosphatase, and CD105. The cells in regenerated pulp-like tissue reacted positively to anti-human mitochondria antibodies, indicating their human origin. This study provides the first evidence showing that pulp-like tissue can be regenerated de novo in emptied root canal space by stem cells from apical papilla and dental pulp stem cells that give rise to odontoblast-like cells producing dentin-like tissue on existing dentinal walls.

The location and characteristics of two populations of dental pulp cells affect tooth development.

This study investigated the characteristics of two dental pulp cell populations during the early stages of crown formation in porcine teeth. A transplantation method was developed to reproduce epithelial cell-mesenchymal cell interactions during odontogenesis (tooth development). The technique allowed two types of cells/tissue to be combined in vivo. Populations of cells localized in the cervical loop epithelium region, dental pulp horn, and dental pulp core chambers were isolated and dissociated into single cells. Each population was examined for its gene-expression pattern using both semiquantitative and quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses, and for its tissue-formation capability by combining the cervical loop epithelial cells with either pulp horn cells or pulp core cells on biodegradable collagen scaffolds that were subsequently examined using histology and immunohistology. Gene-expression patterns showed that pulp horn cells were more mature than pulp core cells. Cervical loop epithelial cells combined with pulp horn cells mainly reconstituted dentin-cementum structures. By contrast, cervical loop epithelial cells combined with pulp core cells reconstituted enamel-dentin structures. These results suggest that mesenchymal cells residing in a specific location of the pulp possess a specific tissue-formation potential when combined with epithelial cells.

Side population cells isolated from porcine dental pulp tissue with self-renewal and multipotency for dentinogenesis, chondrogenesis, adipogenesis, and neurogenesis.

Dental pulp has the potential to form dentin as a regenerative response to caries. This regeneration is mediated by stem/progenitor cells. Thus, stem cell therapy might be of potential utility in induction of reparative dentin. We isolated side population (SP) cells from dental pulp based on the exclusion of the DNA binding dye Hoechst 33342 by flow cytometry and compared its self-renewal capacities and multipotency with non-SP cells and primary pulp cells. The cumulative cell number of the SP cells was greater than the non-SP cells and primary pulp cells. Bmi1 was continuously expressed in SP cells, suggesting longer proliferative lifespan and self-renewal capacity of SP cells. Next, the maintenance of the multilineage differentiation potential of pulp SP cells was investigated. Expression of type II collagen and aggrecan confirmed chondrogenic conversion (30%) of SP cells. SP cells expressed peroxisome proliferator-activated receptor gamma and adaptor protein 2, showing adipogenic conversion. Expression of mRNA and proteins of neurofilament and neuromodulin confirmed neurogenic conversion (90%). These results demonstrate that pulp SP cells maintain multilineage differentiation potential. We further examined whether bone morphogenetic protein 2 (BMP2) could induce differentiation of pulp SP cells into odontoblasts. BMP2 stimulated the expression of dentin sialophosphoprotein (Dspp) and enamelysin in three-dimensional pellet cultures. Autogenous transplantation of the Bmp2-supplemented SP cells on the amputated pulp stimulated the reparative dentin formation. Thus, adult pulp contains SP cells, which are enriched for stem cell properties and useful for cell therapy with BMP2 for dentin regeneration.

Changes in fluoride sensitivity during in vitro senescence of normal human oral cells.

We have previously reported that sodium fluoride (NaF) showed slightly higher cytotoxicity against human oral tumor cell lines than normal human oral cells. Possible changes in the NaF sensitivity of three normal human oral cell types (gingival fibroblast HGF, pulp cell HPC, periodontal ligament fibroblast HPLF) during in vitro ageing were investigated in the present study. When these cells were subcultured at 1:4 split ratio every week, their saturation density declined with increasing population doubling level (PDL), and they ceased to divide when they reached 20 PDL. Mitochondrial function, evaluated by MTT stainability per cell basis, was elevated at the terminal phase. NaF dose-dependently reduced the viable cell number, but did not show any beneficial (growth promoting) effect (so-called "hormesis") at lower concentrations. NaF produced large DNA fragments, without induction of internucleosomal DNA fragmentation, possibly due to weak activation of caspases -3, -8 and -9. Higher concentrations of NaF were required to reduce the number of viable senescent cells than younger cells, indicating that cells become resistant to cytotoxicity of NaF with in vitro ageing.

Tissue engineering of complex tooth structures on biodegradable polymer scaffolds.

Tooth loss due to periodontal disease, dental caries, trauma, or a variety of genetic disorders continues to affect most adults adversely at some time in their lives. A biological tooth substitute that could replace lost teeth would provide a vital alternative to currently available clinical treatments. To pursue this goal, we dissociated porcine third molar tooth buds into single-cell suspensions and seeded them onto biodegradable polymers. After growing in rat hosts for 20 to 30 weeks, recognizable tooth structures formed that contained dentin, odontoblasts, a well-defined pulp chamber, putative Hertwig's root sheath epithelia, putative cementoblasts, and a morphologically correct enamel organ containing fully formed enamel. Our results demonstrate the first successful generation of tooth crowns from dissociated tooth tissues that contain both dentin and enamel, and suggest the presence of epithelial and mesenchymal dental stem cells in porcine third molar tissues.

El efecto en la regeneración tisular guiada del condicionamiento radicular y fibronectina: estudio experimental / The effect in guided tissue regeneration of the root conditioners and fibronectin: experimental study

La utilización de mediadores bioquímicos en la regeneración de tejidos periodontales, abre una interesante perspectiva en el propósito de obtener un mayor procentaje de nueva inserción en relación a la regeneración tisular guiada. En el presente estudio se comparó el efecto de estos mediadores bioquímicos cuando se usan solos o combinados con la técnica de regeneración tisular guiada (QTG). Material y Métodos: 10 perros adultos jóvenes con lesiones periodontales > de 4mm. en los premolares inferiores. Estas lesiones fueron tratadas con RTG más ácido cítrico o tetraciclina y fibronectina en un lado y con cirugía convencional (colgajo) y ácido cítrico o tetraciclina y ficronectina en el lado contrario. Se evaluaron resultados clínicos e histológicos a los 7,15,30,60 y 90 días. Clínicamente a los 90 días se observa menor profundidad del crévice y mayor ganancia de inserción clínica en los casos en que utilizó la RTG en conjunto con los condicionadores rediculares y la fibronectina. Histológicamente también se observa mayor regeneración de tejidos periodontales incluyendo neoformación ósea en la RTG combinada con condicionadores y fibronectina. Como conclusión se puede afirmar que el uso de los mediadores bioquímicos en la regeneración periodontal, es efectivo cuando se combinan con la técnica de RTG