Journal of Dental Research

Craniosynostosis occurs in approximately 1 in 2,000 children and results from the premature fusion of ≥1 cranial sutures. If left untreated, craniosynostosis can cause numerous complications as related to an increase in intracranial pressure or as a direct result from cranial deformities, or both. More than 100 known mutations may cause syndromic craniosynostosis, but the majority of cases are nonsyndromic, occurring as isolated defects. Most cases of craniosynostosis require complex cranial vault reconstruction that is associated with a high risk of morbidity. While the first operation typically has few complications, bone rapidly regrows in up to 40% of children who undergo it. This resynostosis typically requires additional surgical intervention, which can be associated with a high incidence of life-threatening complications. This article reviews work related to the dental and maxillofacial implications of craniosynostosis and discusses clinically relevant animal models related to craniosynostosis and resynostosis. In addition, information is provided on the imaging modalities used to study cranial defects in animals and humans.

Directed acyclic graphs (DAGs) are nonparametric graphical tools used to depict causal relations in the epidemiologic assessment of exposure-outcome associations. Although their use in dental research was first advocated in 2002, DAGs have yet to be widely adopted in this field. DAGs help identify threats to causal inference such as confounders, bias due to subject selection, and inappropriate handling of missing data. DAGs can also inform the data analysis strategy based on relations among variables depicted on it. This article uses the example of a study of temporomandibular disorders (TMDs), investigating causal effects of facial injury on subsequent risk of TMD. We illustrate how DAGs can be used to identify 1) potential confounders, 2) mediators and the consequences of attempt to estimate direct causal effects, 3) colliders and the consequences of conditioning on colliders, and 4) variables that are simultaneously mediators and confounders and the consequences of adjustment for such variables. For example, one DAG shows that statistical adjustment for the pressure pain threshold would necessarily bias the causal relation between facial injury and TMD. Finally, we discuss the usefulness of DAGs during study design, subject selection, and choosing variables to be measured in a study.

Our recent study established the increased circulating microparticles (MPs) and their procoagulant activity in oral squamous cell carcinoma (OSCC). In the present study, we further evaluated different phenotypes of circulating MPs in OSCC patients and explored their clinical significance and effects on angiogenesis (a critical event in tumor progression). To conduct the study, circulating MPs in 45 OSCC patients and 18 healthy volunteers were characterized and quantified by transmission electron microscopy and flow cytometry. Correlations between circulating MPs and clinicopathologic data, microvessel density, and proangiogenic factor levels in patients with OSCC were analyzed by immunohistochemistry and Spearman rank correlation test. Additionally, the in vitro studies were performed with use of human umbilical vein endothelial cells. Our results showed that the levels of circulating MPs as well as the subsets of platelet-derived, endothelium-derived, and pan-leukocyte MPs in stages III to IV OSCC were significantly higher than stages I to II and healthy subjects. Moreover, these increased circulating MPs were significantly correlated with tumor size, TNM stages, microvessel density, and expression levels of vascular endothelial growth factor (VEGF) and matrix metallopeptidase 9 (MMP9) in OSCC patients. The in vitro studies revealed that circulating MPs isolated from OSCC patients could be effectively taken up by human umbilical vein endothelial cells and could promote the proliferation, migration, invasion, and tube formation of recipient endothelial cells, accompanied by increased expression of proangiogenic factors. In summary, circulating MPs play important roles in angiogenesis and local tumor progression of OSCC. Our results shed new light on the progression of OSCC and might be helpful to explore novel treatment strategies targeting tumor angiogenesis.

Systematic reviews have found no evidence to support a benefit of water fluoridation (WF) to prevent dental caries in adult populations. The aim of this natural experiment was to investigate whether lifetime access to fluoridated water is associated with dental caries experience among adults from Florianópolis, Brazil. The data originated from a population-based cohort study (EpiFloripa Adult) initiated in 2009 (n = 1,720) when participants were aged 20 to 59 years. The second wave was carried out in 2012 (n = 1,140) and included a dental examination and a face-to-face questionnaire. Participants residing at the same address since the age of 7 y or before were included in the primary analyses. Sensitivity analyses were also performed. WF was implemented in the city in 2 different periods of time: 1982 (60% of the population) and 1996. Dental caries was assessed by the decayed, missing, and filled teeth (DMFT) index. A combination of residential status, participant’s age, and year of implementation of WF permitted the creation of participants’ lifetime access to fluoridated water: >75%, 50% to 75%, and <50% of a participant’s lifetime. Covariates included sex, age, socioeconomic mobility, educational attainment, income, pattern of dental attendance, and smoking. Participants who accessed fluoridate water <50% of their lifetime presented a higher mean rate ratio of DMFT (1.39; 95% CI, 1.05–1.84) compared with those living >75% of their lifetime with residential access to fluoridated water. Participants living between 50% and 75% and <50% of their lives in fluoridated areas presented a decayed and filled teeth mean ratio of 1.34 (95% CI, 1.02–1.75) and 1.47 (95% CI, 1.05–2.04) higher than those with residential access to fluoridated water >75% of their lifetime, respectively. Longer residential lifetime access to fluoridated water was associated with less dental caries even in a context of multiple exposures to fluoride.

Secondary caries (SC) remains a very important problem with composite restorations. The objectives of this study were to test the acid-buffering ability of several restorative materials and to evaluate whether buffering of the restorative material has an impact on the microbial composition of the biofilm. Disk-shaped specimens of conventional composite, composite with surface prereacted glass-ionomer filler particles (so-called giomer), glass-ionomer cement (GIC), amalgam, and hydroxyapatite (HAp) (control) were exposed to aqueous solutions with pH 4, 5, 6, and 7 and to the medium containing bacteria-produced acids, and pH changes were recorded over several days. Next, material specimens were immersed in bacterial growth medium with pH adjusted to 5. After a 24-h incubation, the extracts were collected and inoculated with a cariogenic (Streptococcus mutans) and a noncariogenic (Streptococcus sanguinis) species. The bacterial growth was monitored both in a single-species model by spectrophotometry and in a dual-species model by viability quantitative polymerase chain reaction. Amalgam and HAp showed the strongest acid-buffering ability, followed by the GIC and the giomer, while the conventional composite did not exhibit any buffering capacity. Furthermore, due to the lack of acid-buffering abilities, composite was not able to increase the pH of the medium (pH 5), which, in the absence of antibacterial properties, allowed the growth of S. mutans, while the growth of S. sanguinis, a less aciduric species, was completely inhibited. A similar effect was observed when bacteria were cultured together: there was a higher percentage of S. mutans and lower percentage of S. sanguinis with the conventional composite than with other materials and HAp. In conclusion, conventional composites lack the ability to increase the local pH, which leads to the outgrowth of more acidogenic/aciduric bacteria and higher cariogenicity of the biofilm. Together with lack of antibacterial properties, lack of buffering may account for the higher susceptibility of composites to SC.

Producing induced pluripotent stem cells (iPSCs) from human tissue for use in personalized medicine strategies or therapeutic testing is at the forefront of medicine. Therefore, identifying a source of cells to reprogram that is easily accessible via a simple noninvasive procedure is of great clinical importance. Reprogramming these cells to iPSCs through nonintegrating methods for genetic manipulation is paramount for regenerative purposes. Here, we demonstrate reprogramming of oral mucosal lamina propria progenitor cells from patients undergoing routine dental treatment. Reprogramming was performed utilizing nonintegrating plasmids containing all 6 pluripotency genes (OCT4, SOX2, KLF4, NANOG, LIN28, and cMYC). Resulting iPSCs lacked genetic integration of the vector genes and had the ability to differentiate down mesoderm, ectoderm, and endoderm lineages, demonstrating pluripotency. In conclusion, oral mucosal lamina propria progenitor cells represent a source of cells that can be obtained with minimal invasion, as they can be taken concurrently with routine treatments. The resulting integration-free iPSCs therefore have great potential for use in personalized medicine strategies.

Our previous study identified that endoplasmic reticulum stress (ERS) plays a critical role in chondrocyte apoptosis and mandibular cartilage thinning in response to compressive mechanical force, although the underlying mechanisms remain elusive. Because the endoplasmic reticulum (ER) is a primary site of intracellular Ca²⁺ storage, we hypothesized that Ca²⁺-dependent ERS might be involved in mechanical stress–mediated mandibular cartilage thinning. In this study, we used in vitro and in vivo models to determine Ca²⁺ concentrations, histological changes, subcellular changes, apoptosis, and the expression of ERS markers in mandibular cartilage and chondrocytes. The results showed that in chondrocytes, cytosolic Ca²⁺ ([Ca²⁺]i) was dramatically increased by compressive mechanical force. Interestingly, the inhibition of Ca²⁺ channels by ryanodine and 2-aminoethoxydiphenyl borate, inhibitors of ryanodine receptors and inositol trisphosphate receptors, respectively, partially rescued mechanical force–mediated mandibular cartilage thinning. Furthermore, chondrocyte apoptosis was also compromised by inhibiting the increase in [Ca²⁺]i that occurred in response to compressive mechanical force. Mechanistically, the ERS induced by compressive mechanical force was also repressed by [Ca²⁺]i inhibition, as demonstrated by a decrease in the expression of the ER stress markers 78 kDa glucose-regulated protein (GRP78) and 94 kDa glucose-regulated protein (GRP94) at both the mRNA and protein levels. Collectively, these data identified [Ca²⁺]i as a critical mediator of the pathological changes that occur in mandibular cartilage under compressive mechanical force and shed light on the treatment of mechanical stress–mediated cartilage degradation.

Cortical excitation responding to periodontal ligament (PDL) stimulation is observed in the rat primary somatosensory (S1), secondary somatosensory, and insular oral region of the cortex (S2/IOR), which are considered to process somatosensation, including nociception. Our previous studies have demonstrated that excitatory propagation induced by PDL stimulation is facilitated in S1 and S2/IOR 1 d after experimental tooth movement (ETM), and tetanic stimulation of IOR induces long-term potentiation of cortical excitatory propagation consistently. These findings raise the possibility that ETM induces neuroplastic changes, and as a result, facilitation of cortical excitation would be sustained for weeks. However, no information is available about the temporal profiles of the facilitated cortical responses. We estimated PDL stimulation-induced cortical excitatory propagation in S1 and S2/IOR of rats by optical imaging 1 to 7 d after ETM of the maxillary first molar. ETM models showed facilitated cortical excitatory propagation in comparison with controls and sham groups 1 d after ETM, but the facilitation gradually recovered to the control level 3 to 7 d after ETM. Sham groups that received wire fixation without orthodontic force tended to enhance cortical responses, although the differences between controls and sham groups were almost insignificant. We also examined the relationship between cortical responses and expression of inflammatory cytokines, interleukin (IL)–1β and tumor necrosis factor (TNF)–α, in PDL of the first molar. The peak amplitude of optical signals responding to PDL stimulation tended to be increased in parallel to the number of IL-1β and TNF-α immunopositive cells, suggesting that, at least in part, the enhancement of cortical responses is induced by PDL inflammation. These findings suggest that ETM-induced facilitation of cortical excitatory propagation responding to PDL stimulation 1 d after ETM recovers to the control level within a week. The time course of the facilitated cortical responses is comparable to that of pain and discomfort induced by clinical orthodontic treatments.

If there is a partial loss of dentin, the exposed dentinal surface should be protected by an indirect pulp capping (IPC) procedure to preserve pulp vitality and prevent symptoms of dentin hypersensitivity. In our previous study, copine7 (CPNE7) induced odontoblast differentiation in vitro and promoted dentin formation in vivo. The aim of this study was to investigate the possibility of IPC therapy using the CPNE7 protein at the exposed dentinal surface and the resulting effects on tertiary dentin formation in a beagle model. CPNE7 promoted mineralization of odontoblasts and had high calcium ion-binding capacity. The in vivo IPC model with canine teeth showed that regeneration of physiologic reactionary dentin with dentinal tubule structures was clearly observed beneath the remaining dentin in the CPNE7 group, whereas irregular features of reparative dentin were generated in the mineral trioxide aggregate (MTA) group. The CPNE7+MTA group also showed typical reactionary dentin without reparative dentin, showing synergistic effects of CPNE7 with MTA. A scanning electron microscopy analysis showed that dentinal tubules beneath the original dentin were occluded by the deposition of peritubular dentin in the CPNE7 and CPNE7+MTA groups, whereas those in the control group were opened. Therefore, CPNE7 may be able to serve as a novel IPC material and improve symptoms of dentin hypersensitivity.

Dentin-pulp regeneration is closely linked to the presence of nerve fibers in the pulp and to the healing mechanism by sprouting of the nerve fiber’s terminal branches beneath the carious injury site. However, little is known about the initial mechanisms regulating this process in carious teeth. It has been recently demonstrated that the complement system activation, which is one of the first immune responses, contributes to tissue regeneration through the local production of anaphylatoxins such as C5a. While few pulp fibroblasts in intact teeth and in untreated fibroblast cultures express the C5a receptor (C5aR), here we show that all dental pulp fibroblasts, localized beneath the carious injury site, do express this receptor. This observation is consistent with our in vitro results, which showed expression of C5aR in lipoteichoic acid–stimulated pulp fibroblasts. The interaction of C5a, produced after complement synthesis and activation from pulp fibroblasts, with the C5aR of these cells mediated the local brain-derived neurotropic factor (BDNF) secretion. Overall, this activation guided the neuronal growth toward the lipoteichoic acid–stimulated fibroblasts. Thus, our findings highlight a new mechanism in one of the initial steps of the dentin-pulp regeneration process, linking pulp fibroblasts to the nerve sprouting through the complement system activation. This may provide a useful future therapeutic tool in targeting the fibroblasts in the dentin-pulp regeneration process.

It is known from the paleontology studies of eutherian mammals that incisor numbers were reduced during evolution. The evolutionary lost incisors may remain as vestigial structures at embryonic stages. The recapitulation of the incisor patterns among mammalian species will potentially uncover the mechanisms underlying the phenotypic transition of incisors during evolution. Here, we showed that a minute tooth formed in the presumptive groove region of the gerbil upper incisor at the early developmental stages, during which multiple epithelial swellings and Shh transcription domains spatiotemporally appeared in the dental epithelium, suggests the existence of vestigial dental primordia. Interestingly, when we trimmed the surrounding mesenchyme from incisor tooth germs at or before the bud stage prior to ex vivo culture, the explants developed different incisor phenotypes ranging from triplicated incisors, duplicated incisors, to Lagomorpha-like incisors, corresponding to the incisor patterns in the eutherian mammals. These results imply that the phenotypic transition of incisors during evolution, as well as the achievement of ultimate incisors in adults, arose from differential integrations of primordia. However, when the incisor tooth germ was trimmed at the cap stage, a grooved incisor developed similar to the normal condition. Furthermore, the incisor tooth germ developed a small but smooth incisor after the additional removal of the minute tooth and a lateral rudiment. These results suggest that multiple dental primordia integrated before the cap stage, with the labial primordia contributing to the labial face of the functional incisor. The minute tooth that occupied the boundary of the 2 labial primordia might be implicated in the groove formation. This study sheds light on how rudiments incorporate into functional organs and aids the understanding of incisor evolution.

Neutrophils exit the vasculature and swarm to sites of inflammation and infection. However, these cells are abundant in the healthy, inflammation-free human oral environment, suggesting a unique immune surveillance role within the periodontium. We hypothesize that neutrophils in the healthy oral cavity occur in an intermediary parainflammatory state that allows them to interact with and contain the oral microflora without eliciting a marked inflammatory response. Based on a high-throughput screen of neutrophil CD (cluster of differentiation) marker expression and a thorough literature review, we developed multicolor flow cytometry panels to determine the surface marker signatures of oral neutrophil subsets in periodontal health and disease. We define here 3 distinct neutrophil subsets: resting/naive circulatory neutrophils, parainflammatory neutrophils found in the healthy oral cavity, and proinflammatory neutrophils found in the oral cavity during chronic periodontal disease. Furthermore, parainflammatory neutrophils manifest as 2 distinct subpopulations—based on size, granularity, and expression of specific CD markers—and exhibit intermediate levels of activation as compared with the proinflammatory oral neutrophils. These intermediately activated parainflammatory populations occur in equal proportions in the healthy oral cavity, with a shift to one highly activated proinflammatory neutrophil population in chronic periodontal disease. This work is the first to identify and characterize oral parainflammatory neutrophils that interact with commensal biofilms without inducing an inflammatory response, thereby demonstrating that not all neutrophils trafficking through periodontal tissues are fully activated. In addition to establishing possible diagnostic and treatment monitoring biomarkers, this oral neutrophil phenotype model builds on existing literature suggesting that the healthy periodontium may be in a parainflammatory state.

Transforming growth factor β (TGFβ) regulates cell proliferation, differentiation, migration, apoptosis, and extracellular matrix production. It also plays a pivotal role in the pathogenesis of gingival overgrowth. Thrombin is a key player in tissue repair, remodeling, and fibrosis after an injury, and it exerts profibrotic effects by activating protease-activated receptors. Connective tissue growth factor (CTGF or CCN2) modulates cell adhesion, migration, proliferation, matrix production, and wound healing. It is overexpressed in many fibrotic disorders, including gingival overgrowth, and it is positively associated with the degree of fibrosis in gingival overgrowth. In human gingival fibroblasts, we previously found that TGFβ1 induced CCN2 protein synthesis through c-jun N-terminal kinase and Smad3 activation. Thrombin stimulates CCN2 synthesis through protease-activated receptor 1 and c-jun N-terminal kinase signaling. Curcumin inhibited TGFβ1- and thrombin-induced CCN2 synthesis. In this study, we demonstrated that thrombin and protease-activated receptor 1 agonist SFLLRN induced latent TGFβ1 activation and Smad3 phosphorylation in human gingival fibroblasts. Pretreatment with a TGFβ-neutralizing antibody, TGFβ type I receptor inhibitor SB431542, and Smad3 inhibitor SIS3 inhibited approximately 86%, 94%, and 100% of thrombin-induced CCN2 synthesis, respectively. Furthermore, blocking integrin subunits αv and β1 with antibodies effectively inhibited SFLLRN-induced Smad3 phosphorylation and CCN2 synthesis and increased activated TGFβ1 levels; however, similar effects were not observed for integrins αvβ3 and αvβ5. These results suggest that protease-activated receptor 1–induced CCN2 synthesis in human gingival fibroblasts is mediated through integrin αvβ1–induced latent TGFβ1 activation and subsequent TGFβ1 signaling. Moreover, curcumin dose dependently decreased thrombin-induced activated TGFβ1 levels. Curcumin-inhibited thrombin-induced CCN2 synthesis in human gingival fibroblasts is caused by the suppression of latent TGFβ1 activation.

The oral microbial community is the best-characterized bacterial ecosystem in the human host. It has been shown in the mouse that oral commensal bacteria significantly contribute to clinically healthy periodontal homeostasis by influencing the number of neutrophils that migrate from the vasculature to the junctional epithelium. Furthermore, in clinically healthy tissue, the neutrophil response to oral commensal bacteria is associated with the select expression of the neutrophil chemokine CXCL2 but not CXCL1. This preliminary study examined the contribution of commensal bacteria on neutrophil location across the tooth/gingival interface. Tissue sections from the root associated mesial (anterior) of the second molar to the root associated distal (posterior) of the second molar were examined for neutrophils and the expression of the neutrophil chemokine ligands CXCL1 and CXCL2. It was found that both the number of neutrophils as well as the expression of CXCL2 but not CXCL1 was significantly increased in tissue sections close to the interdental region, consistent with the notion of select tissue expression patterns for neutrophil chemokine expression and subsequent neutrophil location. Furthermore, mice gavaged with either oral Streptococcus or Lactobacillus sp. bacteria induced a location pattern of neutrophils and CXCL2 expression similar to the normal oral flora. These data indicate for the first time select neutrophil location and chemokine expression patterns associated with clinically healthy tissue. The results reveal an increased inflammatory load upon approaching the interproximal region, which is consistent with the observation that the interproximal region often reveals early clinical signs of periodontal disease.

Nikopensius T, Saag M, Jagomägi T, Annilo T, Kals M, Kivistik PA, Milani L, Metspalu A. 2013. A missense mutation in DUSP6 is associated with Class III malocclusion. J Dent Res. 92(10):893–898. (Original DOI: 10.1177/0022034513502790)

Alikhani M, Lopez JA, Alabdullah H, Vongthongleur T, Sangsuwon C, Alikhani M, Alansari S, Oliveira SM, Nervina JM, Teixeira CC. 2016. High-frequency acceleration: therapeutic tool to preserve bone following tooth extractions. J Dent Res. 95(3):311–318. (Original DOI: 10.1177/0022034515621495)