Salivary annexin A1: A candidate biomarker for periodontitis.

AIM: To compare the salivary proteomic profile of periodontitis-affected (PA) parents and their offspring to periodontally healthy (PH) dyads in the pursuit of possible biomarkers for early diagnosis of this disease. MATERIALS AND METHODS: Unstimulated saliva samples collected from 17 pairs of PA or PH individuals and their children were submitted to mass spectrometric analyses followed by proteomic analyses. Primary PA fibroblasts were triggered towards having an inflammatory response, and an immunoenzymatic assay of its supernatant was performed to validate the obtained data. RESULTS: ANXA1, KRT4, GSTP1, HPX, A2M and KRT13 were lower in PA parents and their children, and IGHG1, CSTB, KRT9, SMR3B, IGHG4 and SERPINA1 were higher. ANXA1 presented the highest fold change, 7.1 times less produced in children of PA parents, and was selected as a potential biomarker for periodontitis. The in vitro assay also showed lower ANXA1 production by cells of PA patients. CONCLUSION: Before any clinical sign of periodontal loss, descendants of PA patients have an altered proteomic profile compared to PH individuals, presenting a lower abundance of ANXA1. This protein is suggested as a potential biomarker for periodontitis.

Obesity Modifies the Proteomic Profile of the Periodontal Ligament.

This study aimed to assess the obesity effects on the proteomic profile of the periodontal ligament of rats submitted to obesity induction by a high-fat diet. Eight Holtzman rats were divided into control (n = 3) and obese (n = 5) groups. The maxillae were histologically processed for laser capture microdissection of the periodontal ligament of the first maxillary molars. Peptide mixtures were analyzed by LC-MS/MS. A total of 1379 proteins were identified in all groups. Among them, 335 (24.30%) were exclusively detected in the obese group, while 129 (9.35%) proteins were uniquely found in the control group. Out of the 110 (7.98%) differentially abundant proteins, 10 were more abundant and 100 had decreased abundance in the obese group. A gene ontology analysis showed some proteins related to obesity in the "extracellular exosome" term among differentially identified proteins in the gene ontology cellular component terms Prelp, Sec13, and Sod2. These three proteins were upregulated in the obese group (p < 0.05), as shown by proteomic and immunohistochemistry analyses. In summary, our study presents novel evidence that the proteomic profile of the periodontal ligament is altered in experimental obesity induction, providing a list of differentially abundant proteins associated with obesity, which indicates that the periodontal ligament is responsive to obesity.

Orthodontic tooth movement alters cementocyte ultrastructure and cellular cementum proteome signature.

Cementum is a mineralized tissue that covers tooth roots and functions in the periodontal attachment complex. Cementocytes, resident cells of cellular cementum, share many characteristics with osteocytes, are mechanoresponsive cells that direct bone remodeling based on changes in loading. We hypothesized that cementocytes play a key role during orthodontic tooth movement (OTM). To test this hypothesis, we used 8-week-old male Wistar rats in a model of OTM for 2, 7, or 14 days (0.5 N), whereas unloaded contralateral teeth served as controls. Tissue and cell responses were analyzed by high-resolution micro-computed tomography, histology, tartrate-resistant acid phosphatase staining for odontoclasts/osteoclasts, and transmission electron microscopy. In addition, laser capture microdissection was used to collect cellular cementum, and extracted proteins were identified by liquid chromatography coupled to tandem mass spectrometry. The OTM model successfully moved first molars mesially more than 250 µm by 14 days introducing apoptosis in a small number of cementocytes and areas of root resorption on mesial and distal aspects. Cementocytes showed increased nuclear size and proportion of euchromatin suggesting cellular activity. Proteomic analysis identified 168 proteins in cellular cementum with 21 proteins found only in OTM sites and 54 proteins only present in control samples. OTM-down-regulated several extracellular matrix proteins, including decorin, biglycan, asporin, and periostin, localized to cementum and PDL by immunostaining. Furthermore, type IV collagen (COL14A1) was the protein most down-regulated (-45-fold) by OTM and immunolocalized to cells at the cementum-dentin junction. Eleven keratins were significantly increased by OTM, and a pan-keratin antibody indicated keratin localization primarily in epithelial remnants of Hertwig's epithelial root sheath. These experiments provide new insights into biological responses of cementocytes and cellular cementum to OTM.

Cementocyte alterations associated with experimentally induced cellular cementum apposition in Hyp mice.

BACKGROUND: Cellular cementum, a mineralized tissue covering apical tooth roots, grows by apposition to maintain the tooth in its occlusal position. We hypothesized that resident cementocytes would show morphological changes in response to cementum apposition, possibly implicating a role in cementum biology. METHODS: Mandibular first molars were induced to super-erupt (EIA) by extraction of maxillary molars, promoting rapid new cementum formation. Tissue and cell responses were analyzed at 6 and/or 21 days post-procedure (dpp). RESULTS: High-resolution micro-computed tomography (micro-CT) and confocal laser scanning microscopy showed increased cellular cementum by 21 dpp. Transmission electron microscopy (TEM) revealed that cementocytes under EIA were 50% larger than control cells, supported by larger pore sizes detected by micro-CT. Cementocytes under EIA displayed ultrastructural changes consistent with increased activity, including increased cytoplasm and nuclear size. We applied EIA to Hyp mutant mice, where cementocytes have perilacunar hypomineralization defects, to test cell and tissue responses in an altered mechanoresponsive milieu. Hyp and WT molars displayed similar super-eruption, with Hyp molars exhibiting 28% increased cellular cementum area versus 22% in WT mice at 21 dpp. Compared to control, Hyp cementocytes featured well-defined, disperse euchromatin and a thick layer of peripherally condensed heterochromatin in nuclei, indicating cellular activity. Immunohistochemistry (IHC) for cementum markers revealed intense dentin matrix protein-1 expression and abnormal osteopontin deposition in Hyp mice. Both WT and Hyp cementocytes expressed gap junction protein, connexin 43. CONCLUSION: This study provides new insights into the EIA model and cementocyte activity in association with new cementum formation.

Comparative proteomic analysis of dental cementum from deciduous and permanent teeth.

BACKGROUND AND OBJECTIVES: Dental cementum (DC) is a mineralized tissue covering tooth roots that plays a critical role in dental attachment. Differences in deciduous vs. permanent tooth DC have not been explored. We hypothesized that proteomic analysis of DC matrix would identify compositional differences in deciduous (DecDC) vs. permanent (PermDC) cementum that might reflect physiological or pathological differences, such as root resorption that is physiological in deciduous teeth but can be pathological in the permanent dentition. METHODS: Protein extracts from deciduous (n = 25) and permanent (n = 12) teeth were pooled (five pools of DecDC, five teeth each; four pools of PermDC, three teeth each). Samples were denatured, and proteins were extracted, reduced, alkylated, digested, and analyzed by liquid chromatography-mass spectrometry (LC-MS/MS). The beta-binomial statistical test was applied to normalized spectrum counts with 5% significance level to determine differentially expressed proteins. Immunohistochemistry was used to validate selected proteins. RESULTS: A total of 510 proteins were identified: 123 (24.1%) exclusive to DecDC; 128 (25.1%) exclusive to PermDC; 259 (50.8%) commonly expressed in both DecDC and PermDC. Out of 60 differentially expressed proteins, 17 (28.3%) were detected in DecDC, including myeloperoxidase (MPO), whereas 43 (71.7%) were detected in PermDC, including decorin (DCN) and osteocalcin (BGLAP). Overall, Gene Ontology (GO) analysis indicated that all expressed proteins were related to GO biological processes that included localization and response to stress, and the GO molecular function of differentially expressed proteins was enriched in cell adhesion, molecular binding, cytoskeletal protein binding, structural molecular activity, and macromolecular complex binding. Immunohistochemistry confirmed the trends for selected differentially expressed proteins in human teeth. CONCLUSIONS: Clear differences were found between the proteomes of DecDC and PermDC. These findings may lead to new insights into developmental differences between DecDC and PermDC, as well as to a better understanding of physiological/pathological events such as root resorption.

Membrane proteome characterization of periodontal ligament cell sets from deciduous and permanent teeth.

BACKGROUND: Physiological roles for the periodontal ligament (PDL) include tooth eruption and anchorage, force absorption, and provision of proprioceptive information. Despite the advances in understanding the biology of PDL cells, there is a lack of information regarding the molecular signature of deciduous (DecPDL) and permanent (PermPDL) PDL tissues. Thus, the present study was designed to characterize the membrane proteome of DecPDL and PermPDL cells. METHODS: Primary PDL cells were obtained (n = 6) and a label-free quantitative proteome of cell membrane-enriched components was performed. Proteome findings were validated by quantitative polymerase chain reaction and Western blot assays in fresh human tissues (n = 8) and primary cell cultures (n = 6). In addition, confocal microscopy was used to verify the expression of target factors in the PDL cell cultures. RESULTS: Comparative gene ontology enrichment analysis evidenced that most stickling differences involved "endomembrane system" (PICALM, STX4, and LRP10), "hydrolase activity" (NCSTN and XRCC6), "protein binding" (PICALM, STX4, GPNMB, VASP, extended-synaptotagmin 2 [ESYT2], and leucine-rich repeat containing 15 [LRRC15]), and "isomerase activity" (FKBP8). Data are available via ProteomeXchange with identifier PXD010226. At the transcript level, high PICALM in DecPDL and ESYT2 and LRRC15 in PermPDL were confirmed in fresh PDL tissues. Furthermore, Western blot analysis confirmed increased levels of PICALM, LRRC15, and ESYT2 in cells and/or fresh tissues, and confocal microscopy confirmed the trends for PICALM and LRRC15 expression in PDL cells. CONCLUSION: We report the first comprehensive characterization of the membrane protein machinery of DecPDL and PermPDL cells, and together, we identified a distinct molecular signature for these cell populations, including unique proteins for DecPDL and PermPDL.

Microproteome of dentoalveolar tissues.

Proteomic analysis of extracellular matrices (ECM) of dentoalveolar tissues can provide insights into developmental, pathological, and reparative processes. However, targeted dissection of mineralized tissues, dental cementum (DC), alveolar bone (AB), and dentin (DE), presents technical difficulties. We demonstrate an approach combining EDTA decalcification and laser capture microdissection (LCM), followed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), to analyze proteome profiles of these tissues. Using the LCM-LC-MS/MS approach, a total of 243 proteins was identified from all tissues, 193 proteins in DC, 147 in AB, and 135 proteins DE. Ninety proteins (37% of total) were common to all tissues, whereas 52 proteins (21%) were overlapping in only two. Also, 101 (42%) proteins were exclusively detected in DC (60), AB (15), or DE (26). Identification in all tissues of expected ECM proteins including collagen alpha-1(I) chain (COL1A1), collagen alpha-1(XII) chain (COL12A1), biglycan (BGN), asporin (ASPN), lumican (LUM), and fibromodulin (FMOD), served to validate the approach. Principal component analysis (PCA) and hierarchical clustering identified a high degree of similarity in DC and AB proteomes, whereas DE presented a distinct dataset. Exclusively and differentially identified proteins were detected from all three tissues. The protein-protein interaction network (interactome) of DC was notable for its inclusion of several indicators of metabolic function (e.g. mitochondrial proteins, protein synthesis, and calcium transport), possibly reflecting cementocyte activity. The DE proteome included known and novel mineralization regulators, including matrix metalloproteinase 20 (MMP-20), 5' nucleotidase (NT5E), and secreted phosphoprotein 24 (SPP-24 or SPP-2). Application of the LCM-LC-MS/MS approach to dentoalveolar tissues would be of value in many experimental designs, including developmental studies of transgenic animals, investigation of treatment effects, and identification of novel regenerative factors.

Secretome Profiling of Periodontal Ligament from Deciduous and Permanent Teeth Reveals a Distinct Expression Pattern of Laminin Chains.

It has been suggested that there are histological and functional distinctions between the periodontal ligament (PDL) of deciduous (DecPDL) and permanent (PermPDL) teeth. Thus, we hypothesized that DecPDL and PermPDL display differences in the constitutive expression of genes/proteins involved with PDL homeostasis. Primary PDL cell cultures were obtained for DecPDL (n = 3) and PermPDL (n = 3) to allow us to perform label-free quantitative secretome analysis. Although a highly similar profile was found between DecPDL and PermPDL cells, comparative secretome analysis evidenced that one of the most stickling differences involved cell adhesion molecules, including laminin subunit gamma 1 (LAMC1) and beta 2 (LAMB2). Next, total RNA and protein extracts were obtained from fresh PDL tissues of deciduous (n = 6) and permanent (n = 6) teeth, and Western blotting and qPCR analysis were used to validate our in vitro findings. Western blot analysis confirmed that LAMC1 was increased in DecPDL fresh tissues (p<0.05). Furthermore, qPCR data analysis revealed that mRNA levels for laminin subunit beta 1 (LAMB1), beta 3 (LAMB3), LAMC1, and gamma 2 (LAMC2) were higher in DecPDL fresh tissues, whereas transcripts for LAMB2 were increased in PermPDL (p<0.05). In conclusion, the differential expression of laminin chains in DecPDL and PermPDL suggests an involvement of laminin-dependent pathways in the control of physiological differences between them.

Global proteome profiling of dental cementum under experimentally-induced apposition.

UNLABELLED: Dental cementum (DC) covers the tooth root and has important functions in tooth attachment and position. DC can be lost to disease, and regeneration is currently unpredictable due to limited understanding of DC formation. This study used a model of experimentally-induced apposition (EIA) in mice to identify proteins associated with new DC formation. Mandibular first molars were induced to super-erupt for 6 and 21days after extracting opposing maxillary molars. Decalcified and formalin-fixed paraffin-embedded mandible sections were prepared for laser capture microdissection. Microdissected protein extracts were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), and the data submitted to repeated measure ANOVA test (RM-ANOVA, alpha=5%). A total of 519 proteins were identified, with 97 (18.6%) proteins found exclusively in EIA sites and 50 (9.6%) proteins exclusively expressed in control sites. Fifty six (10.7%) proteins were differentially regulated by RM-ANOVA (p<0.05), with 24 regulated by the exclusive effect of EIA (12 proteins) or the interaction between EIA and time (12 proteins), including serpin 1a, procollagen C-endopeptidase enhancer, tenascin X (TNX), and asporin (ASPN). In conclusion, proteomic analysis demonstrated significantly altered protein profile in DC under EIA, providing new insights on DC biology and potential candidates for tissue engineering applications. SIGNIFICANCE: Dental cementum (DC) is a mineralized tissue that covers the tooth root surface and has important functions in tooth attachment and position. DC and other periodontal tissues can be lost to disease, and regeneration is currently unpredictable due to lack of understanding of DC formation. This study used a model of experimentally-induced apposition (EIA) in mice to promote new cementum formation, followed by laser capture microdissection (LCM) and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) proteomic analysis. This approach identified proteins associated with new cementum formation that may be targets for promoting cementum regeneration.

EEF1D modulates proliferation and epithelial-mesenchymal transition in oral squamous cell carcinoma.

EEF1D (eukaryotic translation elongation factor 1δ) is a subunit of the elongation factor 1 complex of proteins that mediates the elongation process during protein synthesis via enzymatic delivery of aminoacyl-tRNAs to the ribosome. Although the functions of EEF1D in the translation process are recognized, EEF1D expression was found to be unbalanced in tumours. In the present study, we demonstrate the overexpression of EEF1D in OSCC (oral squamous cell carcinoma), and revealed that EEF1D and protein interaction partners promote the activation of cyclin D1 and vimentin proteins. EEF1D knockdown in OSCC reduced cell proliferation and induced EMT (epithelial-mesenchymal transition) phenotypes, including cell invasion. Taken together, these results define EEF1D as a critical inducer of OSCC proliferation and EMT.

DNA methylation analysis of SOCS1, SOCS3, and LINE-1 in microdissected gingival tissue.

OBJECTIVES: DNA methylation plays a critical role in the regulation of the transcription of the suppressors of cytokine signaling (SOCS) 1 and SOCS3, which are modulators in the inflammation. We hypothesized that the methylation status of SOCS1, SOCS3, and long interspersed nuclear element (LINE)-1 in gingival tissues previously inflamed would be similar to that found in gingival tissues without clinical inflammation in the period studied. MATERIALS AND METHODS: Laser capture microdissection was performed to isolate epithelial and connective gingival tissues. The groups were comprised by ten patients without history of periodontitis and absence of clinical signs of inflammation in the gingiva during the study (healthy group) and ten patients with history of periodontitis, presenting inflammation in the gingival tissue at the first examination of the study (controlled chronic periodontitis group). The gingival biopsies from the controlled chronic periodontitis group were collected after controlling the inflammation. DNA methylation patterns were analyzed using methylation-specific high-resolution melting and combined bisulfite restriction analysis. RESULTS: DNA methylation levels for SOCS1 and SOCS3 did not differ between groups or tissues; likewise, no differences were observed in total LINE-1 methylation or at specific loci. CONCLUSION: At 3 months following control of inflammation in gingival tissues, the methylation profile of SOCS1, SOCS3, and LINE-1 is similar between connective and epithelial tissues from patients that were previously affected or not by chronic inflammation. CLINICAL RELEVANCE: Clinical results of a successful treatment are observed after inflammation control and the molecular findings illustrate local and general methylation patterns in recovering tissues toward health conditions and might help to understand events that are occurring in oral cells.

Characterization of highly osteoblast/cementoblast cell clones from a CD105-enriched periodontal ligament progenitor cell population.

BACKGROUND: It is known that periodontal ligament (PDL) harbors a heterogeneous progenitor cell population at different stages of lineage commitment. However, characterization of PDL stem cells committed to osteoblast/cementoblast (O/C) differentiation remains to be elucidated. The present study is carried out to isolate single cell-derived, cluster of differentiation (CD)105-positive PDL clones and to characterize the clones that present high potential to differentiate toward O/C phenotype in vitro. METHODS: Isolation of single cell-derived colonies (clones) from a CD105-enriched PDL progenitor cell population was performed by the ring-cloning technique. Cell clones were evaluated for their O/C differentiation potential, metabolic activity, and expression of STRO-1 protein. Additionally, the clones that showed potential to O/C differentiation were characterized by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) for expression of runt-related transcriptor factor 2 (RUNX2), alkaline phosphatase, CD105, and CD166 during osteogenic induction. RESULTS: Six PDL-CD105(+) clones were obtained, three being highly O/C clones (C-O) and three others that did not have the ability to produce mineralized matrix in vitro (C-F). The C-O group showed lower metabolic activity compared with the C-F group, and both cell groups were positively immunostained for STRO-1. qRT-PCR analysis demonstrated an increased expression of transcripts for RUNX2 and CD166 during the maturation of C-O cells toward O/C phenotype. CONCLUSIONS: These results provide evidence that PDL-CD105(+) purified progenitor cells comprise a heterogeneous cell population that presents a cell subset with high O/C potential and, further, that surface antigen CD166 is modulated during the O/C maturation of this cell subset.

Proteomic analysis of human dental cementum and alveolar bone.

Dental cementum (DC) is a bone-like tissue covering the tooth root and responsible for attaching the tooth to the alveolar bone (AB) via the periodontal ligament (PDL). Studies have unsuccessfully tried to identify factors specific to DC versus AB, in an effort to better understand DC development and regeneration. The present study aimed to use matched human DC and AB samples (n=7) to generate their proteomes for comparative analysis. Bone samples were harvested from tooth extraction sites, whereas DC samples were obtained from the apical root portion of extracted third molars. Samples were denatured, followed by protein extraction reduction, alkylation and digestion for analysis by nanoAcquity HPLC system and LTQ-FT Ultra. Data analysis demonstrated that a total of 318 proteins were identified in AB and DC. In addition to shared proteins between these tissues, 105 and 83 proteins exclusive to AB or DC were identified, respectively. This is the first report analyzing the proteomic composition of human DC matrix and identifying putative unique and enriched proteins in comparison to alveolar bone. These findings may provide novel insights into developmental differences between DC and AB, and identify candidate biomarkers that may lead to more efficient and predictable therapies for periodontal regeneration. BIOLOGICAL SIGNIFICANCE: Periodontal disease is a highly prevalent disease affecting the world population, which involves breakdown of the tooth supporting tissues, the periodontal ligament, alveolar bone, and dental cementum. The lack of knowledge on specific factors that differentiate alveolar bone and dental cementum limits the development of more efficient and predictable reconstructive therapies. In order to better understand cementum development and potentially identify factors to improve therapeutic outcomes, we took the unique approach of using matched patient samples of dental cementum and alveolar bone to generate and compare a proteome list for each tissue. A potential biomarker for dental cementum was identified, superoxide dismutase 3 (SOD3), which is found in cementum and cementum-associated cells in mouse, pig, and human tissues. These findings may provide novel insights into developmental differences between alveolar bone and dental cementum, and represent the basis for improved and more predictable therapies.

Periosteum-derived cells as an alternative to bone marrow cells for bone tissue engineering around dental implants. A histomorphometric study in beagle dogs.

BACKGROUND: The aim of this study is to investigate the potential use of periosteum-derived cells (PCs) for tissue engineering in peri-implant defects. METHODS: Bone marrow cells (BMCs) and PCs were harvested from seven adult beagle dogs, cultured in vitro, and phenotypically characterized with regard to their osteogenic properties. The animals were then subjected to teeth extraction, and 3 months later, two implant sites were drilled, bone dehiscences created, and dental implants placed. Dehiscences were randomly assigned to one of two groups: PCs (PCs + carrier) and BMCs (BMCs + carrier). After 3 months, the animals were sacrificed and the implants with adjacent hard tissues were processed for undecalcified sections. Bone-to-implant contact, bone fill within the limits of implant threads, and new bone area in a zone lateral to the implant were histometrically obtained. RESULTS: In vitro, phenotypic characterization demonstrated that both cell populations presented osteogenic potential, as identified by the mineral nodule formation and the expression of bone markers. Histometrically, an intergroup analysis showed that both cell-treated defects had similar bone fill within the limits of implant threads and bone-to-implant contact (P >0.05), and although a trend toward higher new bone area values was found for the PC group, there was no significant difference between the experimental groups (P >0.05). CONCLUSIONS: Periosteal and bone marrow cells presented a similar potential for bone reconstruction. As such, periosteum may be considered as an alternative source of osteogenic cells in implant dentistry.

Growth and the modeling/remodeling of the alveolar bone of the rat incisor.

The modeling and remodeling of the rat incisor alveolar bone was followed as the animals grew. The weight of the hemimandible, the length of the socket, and the width of the lower incisor were measured. Osteoclasts and resorption areas were identified by tartrate-resistant acid phosphatase staining. Fluorochrome markers were used to detect and measure osteogenic activities. In the socket related to the periodontal ligament, osteoclasts appeared in scattered sites as well as isolated sites of osteogenic activity, apparently without any variation related to the age of the animals. At the socket facing the dental follicle of young rats, the inner surface was lined with osteoclasts. The number of osteoclasts decreased steadily as the rats grew. In 1-year-old rats, in addition to a few scattered osteoclasts, the internal aspect of the labial wall showed some sites lined with osteoblasts and cement lines indicative of prior bone formation. In young rats, there was a continuous osteogenic activity at the external surface of this wall. The thickness of the labial wall of the socket remained apparently constant; therefore, bone resorption must have occurred at the internal side of the wall. Such osteogenic activity was not observed in old rats. The main forces acting on rat incisors, biting and eruption, are continuous through the life of the animals. Thus, these results indicate that the modeling of the alveolar bone related to the dental follicle, in young rats, can only be associated with another force, specifically, the growth of the incisor.