Real-time simulation of the transplanted tooth using model order reduction.

The biomechanics of transplanted teeth remain poorly understood due to a lack of models. In this context, finite element (FE) analysis has been used to evaluate the influence of occlusal morphology and root form on the biomechanical behavior of the transplanted tooth, but the construction of a FE model is extremely time-consuming. Model order reduction (MOR) techniques have been used in the medical field to reduce computing time, and the present study aimed to develop a reduced model of a transplanted tooth using the higher-order proper generalized decomposition method. The FE model of a previous study was used to learn von Mises root stress, and axial and lateral forces were used to simulate different occlusions between 75 and 175N. The error of the reduced model varied between 0.1% and 5.9% according to the subdomain, and was the highest for the highest lateral forces. The time for the FE simulation varied between 2.3 and 7.2 h. In comparison, the reduced model was built in 17s and interpolation of new results took approximately 2.10-2s. The use of MOR reduced the time for delivering the root stresses by a mean 5.9 h. The biomechanical behavior of a transplanted tooth simulated by FE models was accurately captured with a significant decrease of computing time. Future studies could include using jaw tracking devices for clinical use and the development of more realistic real-time simulations of tooth autotransplantation surgery.

Phenotypic Identification of Dental Pulp Mesenchymal Stem/Stromal Cells Subpopulations with Multiparametric Flow Cytometry.

Dental pulp (DP) is a specialized, highly vascularized, and innervated connective tissue mainly composed of undifferentiated mesenchymal cells, fibroblasts, and highly differentiated dentin-forming odontoblasts. Undifferentiated mesenchymal cells include stem/stromal cell populations usually called dental pulp mesenchymal stem cells (DP-MSCs) which differ in their self-renewal properties, lineage commitment, and differentiation capabilities. Analysis of surface antigens has been largely used to precisely identify these DP-MSC populations. However, a major difficulty is that these antigens are actually not specific for MSCs. Most of the markers used are indeed shared by other cell populations such as progenitor cells, mature fibroblasts, and/or perivascular cells. Accordingly, the detection of only one of these markers in a cell population is clearly insufficient to determine its stemness. Recent data reported that multiparametric flow cytometry, by allowing for the detection of several molecules on the surface of one single cell, is a powerful tool to elucidate the phenotype of a cell population both in vivo and in vitro. So far, DP-MSC populations have been characterized mainly based on the isolated expression of molecules known to be expressed by stem cells, such as Stro-1 antigen, melanoma cell adhesion molecule MCAM/CD146, low-affinity nerve growth factor receptor p75NTR/CD271, and the mesenchymal stem cell antigen MSCA-1. Using multiparametric flow cytometry, we recently showed that human DP-MSCs are indeed phenotypically heterogeneous and form several populations.The present paper describes the multiparametric flow cytometry protocol we routinely use for characterizing DP-MSCs. The description includes the design of the antibody panel and explains the selection of the different parameters related to the data quality control.

Current challenges in human tooth revitalization.

Tooth vitality and health are related to the presence of a living connective tissue, the dental pulp (DP), in the center of the dental organ. The DP contains the tooth immune defence system that is activated against invading oral cariogenic bacteria during the caries process and the tissue repair/regeneration machinery involved following microorganisms' eradication. However, penetration of oral bacteria into the DP often leads to complete tissue destruction and colonization of the endodontic space by microorganisms. Classical endodontic therapies consist of disinfecting then sealing the endodontic space with a gutta percha-based material. However, re-infections of the endodontic space by oral bacteria can occur, owing to the lack of tightness of the material. Recent findings suggest that regenerating a fully functional pulp tissue may be an ideal therapeutic solution to maintain a tooth defence system that will detect and help manage future injuries. The objective of this paper was to explain the different revascularization and regeneration strategies that have been proposed to reconstitute a living DP tissue and to discuss the main challenges that have to be resolved to improve these therapeutic strategies.

Dental Pulp Defence and Repair Mechanisms in Dental Caries.

Dental caries is a chronic infectious disease resulting from the penetration of oral bacteria into the enamel and dentin. Microorganisms subsequently trigger inflammatory responses in the dental pulp. These events can lead to pulp healing if the infection is not too severe following the removal of diseased enamel and dentin tissues and clinical restoration of the tooth. However, chronic inflammation often persists in the pulp despite treatment, inducing permanent loss of normal tissue and reducing innate repair capacities. For complete tooth healing the formation of a reactionary/reparative dentin barrier to distance and protect the pulp from infectious agents and restorative materials is required. Clinical and in vitro experimental data clearly indicate that dentin barrier formation only occurs when pulp inflammation and infection are minimised, thus enabling reestablishment of tissue homeostasis and health. Therefore, promoting the resolution of pulp inflammation may provide a valuable therapeutic opportunity to ensure the sustainability of dental treatments. This paper focusses on key cellular and molecular mechanisms involved in pulp responses to bacteria and in the pulpal transition between caries-induced inflammation and dentinogenic-based repair. We report, using selected examples, different strategies potentially used by odontoblasts and specialized immune cells to combat dentin-invading bacteria in vivo.

Preclinical evidence of potential craniofacial adverse effect of zoledronic acid in pediatric patients with bone malignancies.

High doses of zoledronic acid (ZOL), one of the most potent inhibitors of bone resorption, are currently evaluated in phase III clinical trials in Europe for the treatment of malignant pediatric primary bone tumors. The impact of such an intensive treatment on the craniofacial skeleton growth is a critical question in the context of patients with actively growing skeleton; in particular, in light of our previous studies evidencing that endochondral bone formation was transiently disturbed by high doses of ZOL. Two protocols adapted from pediatric treatments were developed for newborn mice (a total of 5 or 10 injections of ZOL 50µg/kg every two days). Their impact on skull bones and teeth growth was analyzed by X-rays, microCT and histology up to 3months after the last injection. ZOL administrations induced a transient delay of skull bone growth and an irreversible delay in incisor, first molar eruption and root elongation. Other teeth were affected, but most were erupted by 3months. Root histogenesis was severely impacted for all molars and massive odontogenic tumor-like structures were observed in all mandibular incisors. High doses of ZOL irreversibly disturbed teeth eruption and elongation, and delayed skull bone formation. These preclinical observations are essential for the follow-up of onco-pediatric patients treated with ZOL.

Human dental pulp stem cells cultured in serum-free supplemented medium.

UNLABELLED: Growing evidence show that human dental pulp stem cells (DPSCs) could provide a source of adult stem cells for the treatment of neurodegenerative pathologies. In this study, DPSCs were expanded and cultured with a protocol generally used for the culture of neural stem/progenitor cells. METHODOLOGY: DPSC cultures were established from third molars. The pulp tissue was enzymatically digested and cultured in serum-supplemented basal medium for 12 h. Adherent (ADH) and non-adherent (non-ADH) cell populations were separated according to their differential adhesion to plastic and then cultured in serum-free defined N2 medium with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Both ADH and non-ADH populations were analyzed by FACS and/or PCR. RESULTS: FACS analysis of ADH-DPSCs revealed the expression of the mesenchymal cell marker CD90, the neuronal marker CD56, the transferrin receptor CD71, and the chemokine receptor CXCR3, whereas hematopoietic stem cells markers CD45, CD133, and CD34 were not expressed. ADH-DPSCs expressed transcripts coding for the Nestin gene, whereas expression levels of genes coding for the neuronal markers ß-III tubulin and NF-M, and the oligodendrocyte marker PLP-1 were donor dependent. ADH-DPSCs did not express the transcripts for GFAP, an astrocyte marker. Cells of the non-ADH population that grew as spheroids expressed Nestin, ß-III tubulin, NF-M and PLP-1 transcripts. DPSCs that migrated out of the spheroids exhibited an odontoblast-like morphology and expressed a higher level of DSPP and osteocalcin transcripts than ADH-DPSCs. CONCLUSION: Collectively, these data indicate that human DPSCs can be expanded and cultured in serum-free supplemented medium with EGF and bFGF. ADH-DPSCs and non-ADH populations contained neuronal and/or oligodendrocyte progenitors at different stages of commitment and, interestingly, cells from spheroid structures seem to be more engaged into the odontoblastic lineage than the ADH-DPSCs.

Lipopolysaccharide-binding protein inhibits toll-like receptor 2 activation by lipoteichoic acid in human odontoblast-like cells.

INTRODUCTION: Previous studies have suggested that odontoblasts sense gram-positive bacteria components through Toll-like receptor 2 (TLR2) and trigger dental pulp immunity by producing proinflammatory cytokines. Currently, the factors that modulate odontoblast TLR2 activation are unknown. Our aim was to investigate lipopolysaccharide-binding protein (LBP) effects on the TLR2-mediated odontoblast response. METHODS: Human odontoblast-like cells were stimulated with lipoteichoic acid (LTA) (a TLR2 ligand), LBP, CD14 (a TLR2 cofactor), or various combinations of LTA/LBP, LTA/CD14, or LTA/CD14/LBP. CXCL8, IL6, and TLR2 gene expression was assessed by real-time polymerase chain reaction. CXCL8 and interleukin (IL)-6 production was determined by enzyme-linked immunosorbent assay in culture supernatants of cells stimulated with LTA, LTA/CD14, or LTA/CD14/LBP. LBP effects on nuclear factor kappa B (NF-κB), p38, JNK, ERK, STAT3, and p70S6 signaling pathways were determined in LTA-stimulated odontoblast-like cells with a multiplex biometric immunoassay. LBP effects were compared with specific inhibitors of these signaling pathways. LBP transcript and protein were investigated in vivo in healthy and inflamed dental pulps by real-time polymerase chain reaction and immunohistochemistry. RESULTS: Activation of CXCL8, IL6, and TLR2 gene expression and CXCL8 and IL-6 secretion in LTA- and LTA/CD14-stimulated odontoblast-like cells was significantly decreased by LBP. LBP inhibited NF-κB and p38 signaling pathways in LTA-stimulated cells in a similar way to NF-κB and p38 inhibitors. LBP transcript and protein were detected in vivo in inflamed dental pulps but not in healthy ones. CONCLUSIONS: These results demonstrate that LBP reduces TLR2-dependent production of inflammatory cytokines by odontoblast-like cells. We suggest that in this way it could modulate host defense in human dental pulp.

Surgical and prosthetic treatment in an elderly patient affected by unilateral idiopathic gingival fibromatosis: a case report.

OBJECTIVES: The aim was to present the diagnosis and treatment of a case of unilateral idiopathic gingival fibromatosis (IGF) in a geriatric patient. BACKGROUND: IGF is a rare condition characterised by an enlargement of the attached and marginal gingivae with no obvious association to any causative factor. Gingival overgrowth causes inaesthetic changes and clinical symptoms such as speech disturbances, tooth movement, and occlusal problems. MATERIALS AND METHODS: A 65-year-old female presented localised enlargement of mandibular gingiva, malpositioning of anterior mandibular teeth, and difficulty in speech and mastication. IGF also causes numerous aesthetic and psychological problems. Treatment consisted of multiple extractions, surgical therapy, and early prosthetic rehabilitation to restore function and appearance and to prevent recurrence. RESULTS: Excellent aesthetic result and psychological benefit were achieved, and no fibromatosis recurrence was detected after 1 year following surgery. CONCLUSION: Gingival resection of the tissue excess and early oral rehabilitation with removable dentures have so far greatly improved patient's quality of life.

Expression of NOD2 is increased in inflamed human dental pulps and lipoteichoic acid-stimulated odontoblast-like cells.

Human odontoblasts trigger immune response s to oral bacteria that invade dental tissues during the caries process. To date, their ability to regulate the expression of the nucleotide-binding domain leucine-rich repeat containing receptor NOD2 when challenged by Gram-positive bacteria is unknown. In this study, we investigated NOD2 expression in healthy and inflamed human dental pulps challenged by bacteria, and in cultured odontoblast-like cells stimulated with lipoteichoic acid (LTA), a Toll-like receptor (TLR) 2 agonist which is specific for Gram-positive bacteria. We found that NOD2 gene expression was significantly up-regulated in pulps with acute inflammation compared to healthy ones. In vitro, LTA augmented NOD2 gene expression and protein level in odontoblast-like cells. The increase was more pronounced in odontoblast-like cells compared to dental pulp fibroblasts. Blocking experiments in odontoblast-like cells with anti-TLR2 antibody strongly reduced the NOD2 gene expression increase, whereas stimulation with the synthetic TLR2 ligand Pam(2)CSK(4) confirmed NOD2 gene up-regulation following TLR2 engagement. These data suggest that NOD2 up-regulation is part of the odontoblast immune response to Gram-positive bacteria and might be important in protecting human dental pulp from the deleterious effects of cariogenic pathogens.

Toll-like receptor 2 activation by lipoteichoic acid induces differential production of pro-inflammatory cytokines in human odontoblasts, dental pulp fibroblasts and immature dendritic cells.

Odontoblasts, dental pulp fibroblasts and immature dendritic cells (DCs) have been involved in the human dental pulp immune response to oral pathogens that invade dentine during the caries process. How they regulate the inflammatory response to Gram-positive bacteria remains nevertheless largely unknown. In this study we investigated the production of the pro-inflammatory cytokines tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-8 (CXCL8) in these three cell types upon stimulation with lipoteichoic acid (LTA), a cell wall component of Gram-positive bacteria that activates the pattern recognition molecule Toll-like receptor 2 (TLR2). We observed that TNF-alpha gene expression was up-regulated in all LTA-stimulated cell types. IL-1beta gene expression was not or barely detectable in odontoblast-like cells and pulp fibroblasts when stimulated or not, but was expressed in immature DCs and increased upon stimulation. TNF-alpha and IL-1beta proteins were detected in DC culture supernatants but not in odontoblast-like cell and pulp fibroblast ones. CXCL8 gene and protein were clearly expressed and increased in the three cell types upon LTA stimulation. These data indicate that LTA-dependent TLR2 activation in odontoblasts and pulp fibroblasts, in contrast to immature DCs, does not lead to significant TNF-alpha and IL-1beta production, but that all three cell types influence the pulp inflammatory/immune response through CXCL8 synthesis and secretion.

A clinical and histological study of dental defects in a 10-year-old girl with pseudoxanthoma elasticum and amelogenesis imperfecta.

BACKGROUND: The prominent dental feature of a 10-year-old girl was severely hypoplastic enamel in permanent teeth. CASE REPORT: Severe dental defects were detected in a 10-year-old female patient affected by pseudoxanthoma elasticum and amelogenesis imperfecta. An orthopantomographic examination revealed a reduction of enamel thickness on the crown of all erupted and unerupted teeth, agenesis of the maxillary right second premolar, delayed eruption of mandibular first premolars, and the presence of large calcifications in all tooth pulp chambers. A detailed histological analysis of permanent mandibular first molars showed that pulp calcifications presented a concentric laminate organization and merged to almost completely obliterate the pulp chamber. Osteodentine was visible all along the pulpal surface of the radicular dentine. Broad resorption areas were present in the outermost dentine at both coronal and radicular levels. Radicular resorption areas presented a typical rectangular form and were filled with acellular cementum. Cementum thickness was highly increased on the root surface. Apposition of cellular cementum-like tissue was also observed on the coronal dentine surface. CONCLUSION: Before treating patients affected by amelogenesis imperfecta and/or pseudoxanthoma elasticum, paediatric dentists should be aware of the presence of pulp calcifications that add to the complexity of endodontic procedures.

Human dental follicle cells acquire cementoblast features under stimulation by BMP-2/-7 and enamel matrix derivatives (EMD) in vitro.

The dental follicle (DF) surrounding the developing tooth germ is an ectomesenchymal tissue composed of various cell populations derived from the cranial neural crest. Human dental follicle cells (HDFC) are believed to contain precursor cells for cementoblasts, periodontal ligament cells, and osteoblasts. Bone morphogenetic proteins (BMPs) produced by Hertwig's epithelial root sheath or present in enamel matrix derivatives (EMD) seem to be involved in the control of DF cell differentiation, but their precise function remains largely unknown. We report the immunolocalization of STRO-1 (a marker of multipotential mesenchymal progenitor cells) and BMP receptors (BMPR) in DF in vivo. In culture, HDFC co-express STRO-1/BMPR and exhibit multilineage properties. Incubation with rhBMP-2 and rhBMP-7 or EMD for 24 h increases the expression of BMP-2 and BMP-7 by HDFC. Long-term stimulation of these cells by rhBMP-2 and/or rhBMP-7 or EMD significantly increases alkaline phosphatase activity (AP) and mineralization. Expression of cementum attachment protein (CAP) and cementum protein-23 (CP-23), two putative cementoblast markers, has been detected in EMD-stimulated whole DF and in cultured HDFC stimulated with EMD or BMP-2 and BMP-7. RhNoggin, a BMP antagonist, abolishes AP activity, mineralization, and CAP/CP-23 expression in HDFC cultures and the expression of BMP-2 and BMP-7 induced by EMD. Phosphorylation of Smad-1 and MAPK is stimulated by EMD or rhBMP-2. However, rhNoggin blocks only Smad-1 phosphorylation under these conditions. Thus, EMD may activate HDFC toward the cementoblastic phenotype, an effect mainly (but not exclusively) involving both exogenous and endogenous BMP-dependent pathways.

Expression of the TGF-beta/BMP inhibitor EVI1 in human dental pulp cells.

Members of the TGF-beta/BMP family of growth factors induce odontoblast differentiation and reparative dentin synthesis, and their use has been proposed to stimulate pulp healing during dental therapeutics in human. However, factors that modulate TGF-beta and/or BMP signalling during odontoblast differentiation and physiology remain largely unknown. To identify them, we compared expression profiles of TGF-beta/BMP-related genes in pulp fibroblast- and odontoblast-like cells cultured from human dental pulp explants using cDNA gene arrays. We evidenced that the gene encoding ecotropic viral integration site-1 (EVI1), a transcription factor that inhibits TGF-beta/BMP signalling, was under-expressed in odontoblast-like cells. This result was verified by real-time PCR and, at the protein level, by immunohistochemistry. In vivo, real-time PCR analysis revealed that EVI1 was expressed in the dental pulp, at a level similar to brain, but lower than in lung, kidney or trachea. The protein was localized in dental pulp samples in pulp core and subodontoblast cells. Staining intensity progressively decreased from the radicular to the coronal pulp where EVI1 staining was almost undetectable in odontoblasts. Our data suggest that fine regulation of the EVI1 level in the human dental pulp might be important in the TGF-beta/BMP-induced modulation of dental pulp cell kinetics and/or odontoblast differentiation.