*This article is free to read on the publisher's website*

BACKGROUND:
Over the past 10 years, the use of saliva as a diagnostic fluid has gained attention and has become a translational research success story. Some of the current nanotechnologies have been demonstrated to have the analytical sensitivity required for the use of saliva as a diagnostic medium to detect and predict disease progression. However, these technologies have not yet been integrated into current clinical practice and work flow.

CONTENT:
As a diagnostic fluid, saliva offers advantages over serum because it can be collected noninvasively by individuals with modest training, and it offers a cost-effective approach for the screening of large populations. Gland-specific saliva can also be used for diagnosis of pathology specific to one of the major salivary glands. There is minimal risk of contracting infections during saliva collection, and saliva can be used in clinically challenging situations, such as obtaining samples from children or handicapped or anxious patients, in whom blood sampling could be a difficult act to perform. In this review we highlight the production of and secretion of saliva, the salivary proteome, transportation of biomolecules from blood capillaries to salivary glands, and the diagnostic potential of saliva for use in detection of cardiovascular disease and oral and breast cancers. We also highlight the barriers to application of saliva testing and its advancement in clinical settings.

SUMMARY:
Saliva has the potential to become a first-line diagnostic sample of choice owing to the advancements in detection technologies coupled with combinations of biomolecules with clinical relevance. (C) 2011 American Association for Clinical Chemistry

Diagnostic potential of saliva : current state and future applications

Early childhood caries (ECC) is a term used to describe dental caries in children aged 6 years or younger. Oral streptococci, such as Streptococcus mutans and Streptococcus sorbrinus, are considered to be the main etiological agents of tooth decay in children. Other bacteria, such as Prevotella spp. and Lactobacillus spp., and fungus, that is, Candida albicans, are related to the development and progression of ECC. Biomolecules in saliva, mainly proteins, affect the survival of oral microorganisms by multiple innate defensive mechanisms, thus modulating the oral microflora. Therefore, the protein composition of saliva can be a sensitive indicator for dental health. Resistance or susceptibility to caries may be significantly correlated with alterations in salivary protein components. Some oral microorganisms and saliva proteins may serve as useful biomarkers in predicting the risk and prognosis of caries. Current research has generated abundant information that contributes to a better understanding of the roles of microorganisms and salivary proteins in ECC occurrence and prevention. This review summarizes the microorganisms that cause caries and tooth-protective salivary proteins with their potential as functional biomarkers for ECC risk assessment. The identification of biomarkers for children at high risk of ECC is not only critical for early diagnosis but also important for preventing and treating the disease.

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Salivary proteins and microbiota as biomarkers for early childhood caries risk assessment

Tooth wear had an adverse effect on several aspects of patient satisfaction.

Impact of tooth wear on daily living

The Global Burden of Oral Diseases affects 3.5 billion people worldwide, representing the number of people affected by the burden of untreated dental caries, severe periodontal disease, and edentulism. Thus, much more efforts in terms of diagnostics and treatments must be provided in the fight of these outcomes. In this sense, recently, the study of saliva as biological matrix has been identified as a new landmark initiative in the search of novel and useful biomarkers to prevent and diagnose these conditions. Specifically, saliva is a rich reservoir of different proteins and peptides and accessible due to recent advances in molecular biology and specially in targeted and unbiased proteomics technologies. Nonetheless, emerging barriers are an obstacle to the study of the salivary proteome in an effective way. This review aims at giving an overall perspective of salivary biomarkers identified in several oral diseases by means of molecular biology approaches.

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Protein-Based Salivary Profiles as Novel Biomarkers for Oral Diseases.

Dental follicle cells (DFCs) are a group of mesenchymal progenitor cells surrounding the tooth germ, responsible for cementum, periodontal ligament, and alveolar bone formation in tooth development. Cascades of signaling pathways and transcriptional factors in DFCs are involved in directing tooth eruption and tooth root morphogenesis. Substantial researches have been made to decipher multiple aspects of DFCs, including multilineage differentiation, senescence, and immunomodulatory ability. DFCs were proved to be multipotent progenitors with decent amplification, immunosuppressed and acquisition ability. They are able to differentiate into osteoblasts/cementoblasts, adipocytes, neuron-like cells, and so forth. The excellent properties of DFCs facilitated clinical application, as exemplified by bone tissue engineering, tooth root regeneration, and periodontium regeneration. Except for the oral and maxillofacial regeneration, DFCs were also expected to be applied in other tissues such as spinal cord defects (SCD), cardiomyocyte destruction. This article reviewed roles of DFCs in tooth development, their properties, and clinical application potentials, thus providing a novel guidance for tissue engineering.

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Dental Follicle Cells: Roles in Development and Beyond.

Severe early childhood caries (s-ECC), which has quite high prevalence among children, is a widespread problem with significant impacts among both developing and developed countries. At present, it is widely known that no early detective techniques and diagnostic tests could have high sensitivity and specificity when using for clinical screening of s-ECC. In this study, we had applied magnetic bead (MB)-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to screen distinctive candidate biomarkers of this disease, so as to establish protein profiles and diagnostic models of s-ECC. Firstly, we used the technique mentioned above to detect specifically expressed peptides in saliva samples from ten children with s-ECC, separately at the time point of before, 1 and 4 weeks after dental treatment. Then a diagnostic model for s-ECC was established with the K nearest-neighbour method, which was validated in another six children in the next stage of study. After that, linear ion trap-orbitrap-mass spectrometry (LTQ-Orbitrap-MS) was performed to identify which of the proteins in saliva might be the origination of these peptides. We found that seven peptide peaks were significantly different when comparing the three time points, among them two were higher, while other five were lower in the pre-treatment s-ECC group compared with post-treatment. The sensitivity and specificity of the diagnostic model we built were both 83.3 %. Two of these peptides were identified to be segments of histatin-1, which was one important secretory protein in saliva. Hereby we confirmed that MB-based MALDI-TOF MS is an effective method for screening distinctive peptides from the saliva of junior patients with s-ECC, and histatin-1 may probably be one important candidate biomarker of this common dental disease. These findings might have bright prospect in future in establishing new diagnostic methods for s-ECC.

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Salivary peptidome profiling for diagnosis of severe early childhood caries

Cleidocranial dysplasia (CCD) is an autosomal dominant inheritable skeletal disorder characterized by cranial dysplasia, clavicle hypoplasia and dental abnormalities. This disease is mainly caused by heterozygous mutations in RUNX2, a gene that encodes an osteoblast-specific transcription factor. In the present study, mutational analyses of RUNX2 gene were performed on four unrelated Chinese patients with CCD. Four different RUNX2 mutations were detected in these patients, including one nonsense mutation (c.199C>T p.Q67X) and three missense mutations (c.338T>G p.L113R, c.557G>C p.R186T and c.673C>T p.R225W). Among them, two mutations (c.199C>T p.Q67X and c.557G>C p.R186T) were novel and the other two had been reported in previous literatures. Except for Q67X mutation located in the Q/A domain, other three mutations were clustered within the highly conserved Runt domain. Green fluorescent protein (GFP) and RUNX2 fusion protein analyses in vitro showed that nuclear accumulation of RUNX2 protein was disturbed by Q67X mutation, while the other two mutations (c.338T>G p.L113R and c.557G>C p.R186T) had no effects on the subcellular distribution of RUNX2. Luciferase reporter assay demonstrated that all the three novel RUNX2 mutations significantly reduced the transactivation activity of RUNX2 on osteocalcin promoter. Our findings enrich the evidence of molecular genetics that the mutations of RUNX2 gene are responsible for CCD.

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Analysis of novel RUNX2 mutations in Chinese patients with cleidocranial dysplasia.

The aim of this study was to evaluate the epidemiology of tooth wear in Beijing and to establish appropriate preventive measures. This cross-sectional analysis involved a questionnaire survey conducted for 1,812 individuals aged 12–74 years in Beijing. Subjects were local residents living in the region for >6 months before the survey. Subjects were evaluated using clinical examinations with the basic erosive wear examination index and a self-administered questionnaire. Statistical analyses were performed using SPSS software. The prevalence of maxillary tooth wear was 84.9% for the molars, 68.9% for the premolars, 74.1% for the canines and 97% for the incisors. In the mandible, the corresponding prevalence rates were 85.2%, 59.3%, 78.6% and 97.4%, respectively. The occlusal, incisal and cervical surfaces showed more frequent wear compared with the other surfaces. Age, acidic beverages, xerostomia and brushing habits were identified as risk factors for tooth wear (P<0.05). Tooth wear is common in Beijing. Specific preventive measures should be recommended for individuals reporting excessive consumption of fruits and/or acidic beverages, and those with xerostomia. In particular, incisor wear should be carefully monitored in individuals of all age groups.

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Tooth wear: a cross-sectional investigation of the prevalence and risk factors in Beijing, China.

Evaluation of root and alveolar bone development of unilateral osseous impacted immature maxillary central incisors after the closed-eruption technique.

Disturbed permanent tooth eruption is common in cleidocranial dysplasia (CCD), a skeletal disorder caused by heterozygous mutation of RUNX2, but the mechanism underlying is still unclear. As it is well known that dental follicle cells (DFCs) play a critical role in tooth eruption, the changed biological characteristics of DFCs might give rise to disturbance of permanent tooth eruption in CCD patients. Thus, primary DFCs from one CCD patient and normal controls were collected to investigate the effect of RUNX2 mutation on the bone remodeling activity of DFCs and explore the mechanism of impaired permanent tooth eruption in this disease. Conservation and secondary structure analysis revealed that the RUNX2 mutation (c.514delT, p.172fs) found in the present CCD patient was located in the highly conserved RUNT domain and converted the structure of RUNX2. After osteogenic induction, we found that the mineralised capacity of DFCs and the expression of osteoblast-related genes, including RUNX2, ALP, OSX, OCN and Col Iα1, in DFCs was severely interfered by the RUNX2 mutation found in CCD patients. To investigate whether the osteogenic deficiency of DFCs from the CCD patient can be rescued by RUNX2 restoration, we performed 'rescue' experiments. Surprisingly, the osteogenic deficiency and the abnormal expression of osteoblast-associated genes in DFCs from the CCD patient were almost rescued by overexpression of wild-type RUNX2 using lentivirus. All these findings indicate that RUNX2 mutation can reduce the osteogenic capacity of DFCs through inhibiting osteoblast-associated genes, thereby disturbing alveolar bone formation, which serves as a motive force for tooth eruption. This effect may provide valuable explanations and implications for the mechanism of delayed permanent tooth eruption in CCD patients.

Xiaozhe Wang

Papers

Salivary peptidome profiling for diagnosis of severe early childhood caries

Analysis of novel RUNX2 mutations in Chinese patients with cleidocranial dysplasia.

Tooth wear: a cross-sectional investigation of the prevalence and risk factors in Beijing, China.

Evaluation of root and alveolar bone development of unilateral osseous impacted immature maxillary central incisors after the closed-eruption technique.

RUNX2 mutation reduces osteogenic differentiation of dental follicle cells in cleidocranial dysplasia.