RESUMO
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.
Assuntos
Cemento Dentário , Dentição Permanente , Cromatografia Líquida , Humanos , Proteômica , Espectrometria de Massas em Tandem , Dente DecíduoRESUMO
Bone sialoprotein (BSP) is a multifunctional extracellular matrix (ECM) protein present in bone and cementum. Global in vivo ablation of BSP leads to bone mineralization defects, lack of acellular cementum, and periodontal breakdown. BSP harbors three main functional domains: N-terminal collagen-binding domain, hydroxyapatite-nucleating domain, and C-terminal RGD integrin-binding signaling domain. How each of these domains contributes to BSP function(s) is not understood. We hypothesized that collagen-binding and RGD domains play distinct roles in cementoblast functions. Three CRISPR/Cas9 gene-edited cell lines were derived from control wild-type (WT) OCCM.30 murine immortalized cementoblasts: 1) deletion of the N-terminus of BSP after signal peptide, including entire collagen binding domain (Ibsp∆N-Term); 2) deletion of exon 4 (majority of collagen-binding domain; Ibsp∆Ex4); and 3) deletion of C-terminus of BSP including the integrin binding RGD domain (Ibsp∆C-Term). Compared to WT, Ibsp∆Ex4 and Ibsp∆C-Term cell lines showed reduced BSP secretion, in vitro. Abnormal cell morphology was observed in all mutant cell lines, with Ibsp∆C-Term showing highly disorganized cytoskeleton. All mutant cell lines showed significantly lower cell proliferation compared to WT at all timepoints. Ibsp∆N-Term cells showed reduced cell migration by 24 h. All mutants exhibited over 50 % significant reduced mineralization at days 6 and 10. While WT cells were largely unaffected by seeding density, mutant cells failed to mineralize at lower cell density. Mutant cell lines diverged from WT and from each other by dysregulated expression in 23 genes involved in mineralization, ECM, and cell signaling. In summary, disabling BSP functional domains led to profound and distinct changes in cementoblast cell functions, especially dysregulated gene expression and reduced mineralization, in a way did not align with a straightforward narrative where each functional domain caused specific, expected differences. Instead, the study uncovered a significant level of complexity in how different mutant forms of BSP affected cell functions, in vitro.
Assuntos
Cemento Dentário , Proteínas da Matriz Extracelular , Camundongos , Animais , Sialoproteína de Ligação à Integrina/genética , Sialoproteína de Ligação à Integrina/metabolismo , Cemento Dentário/metabolismo , Colágeno , Integrinas , OligopeptídeosRESUMO
BACKGROUND: Cellular cementum (CC) includes cementocytes, cells suspected to regulate CC formation or resorption as osteocytes do in bone. Sclerostin (SOST) is a secreted negative regulator of Wnt/ß-catenin signaling expressed by osteocytes and cementocytes. Osteocyte SOST expression reduces bone formation. We investigated the functional importance of SOST in CC compared with alveolar bone (AB) using a Sost knockout (Sost-/-) mouse model to better understand the role of cementocytes in CC. METHODS: Mandibles and femurs of Sost-/- and wild-type (WT) mice were analyzed at 42 and 120 days postnatal (dpn). Maxillary first molars were bilaterally extracted at 42 dpn and both AB healing (maxillary molar sockets) and CC apposition (mandibular first molars) were examined at 21 days post-procedure. Analyses included micro-computed tomography, histology, and immunohistochemistry. RESULTS: Femur cortical and trabecular bone and mandibular bone volumes were similarly increased in Sost-/- versus WT mice at 42 and/or 120 dpn. In contrast to previous reports, CC was not increased by Sost-/- at either age. We conducted challenge experiments on AB and CC to explore tissue-specific responses. Post-extraction AB healing was improved by Sost deletion. In contrast, experimentally-induced apposition in molars failed to stimulate increased CC formation in Sost-/- versus WT mice. Wnt pathway markers AXIN2 and DKK1, which were increased in Sost-/- versus WT AB osteocytes, were unchanged in cementocytes. CONCLUSIONS: These data indicate CC is less responsive than AB to SOST deletion. Within the study limitations, these results do not support cementocytes as critical for directing increased CC formation. PLAIN LANGUAGE SUMMARY: Sclerostin is a protein known to inhibit bone formation, and removing sclerostin leads to more bone formation. Cementum is the thin layer that covers the surface of the tooth's root. Previous studies suggest that inhibiting sclerostin can similarly increase the amount of cementum. We wanted to compare the response of cementum and bone when sclerostin is absent to understand similarities and differences between these two tissues. In this study, we removed the Sost gene (the gene which produces sclerostin) in mice. We found that mice without sclerostin have more bone in their legs and jaws. Moreover, mice without sclerostin also healed better after tooth removal compared with normal mice. Surprisingly, unlike previous studies, we found that the amount of cementum was not different in mice without sclerostin compared with normal mice. Additionally, we challenged the cementum by taking out the opposing tooth to cause the first mandibular molar to move up by building more cementum. Even with this challenge, we found no difference in the amount of cementum in mice lacking sclerostin compared with normal mice. Therefore, we conclude here that cementum is less sensitive to the absence of sclerostin compared with bone.
RESUMO
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.
Assuntos
Proteoma , Técnicas de Movimentação Dentária , Animais , Cemento Dentário , Masculino , Osteoclastos , Proteômica , Ratos , Ratos Wistar , Raiz Dentária , Microtomografia por Raio-XRESUMO
BACKGROUND: Matrix metallopeptidase 20 (MMP20) is an evolutionarily conserved protease that is essential for processing enamel matrix proteins during dental enamel formation. MMP20 mutations cause human autosomal recessive pigmented hypomaturation-type amelogenesis imperfecta (AI2A2; OMIM #612529). MMP20 is expressed in both odontoblasts and ameloblasts, but its function during dentinogenesis is unclear. METHODS: We characterized 10 AI kindreds with MMP20 defects, characterized human third molars and/or Mmp20-/- mice by histology, Backscattered Scanning Electron Microscopy (bSEM), µCT, and nanohardness testing. RESULTS: We identified six novel MMP20 disease-causing mutations. Four pathogenic variants were associated with exons encoding the MMP20 hemopexin-like (PEX) domain, suggesting a necessary regulatory function. Mutant human enamel hardness was softest (13% of normal) midway between the dentinoenamel junction (DEJ) and the enamel surface. bSEM and µCT analyses of the third molars revealed reduced mineral density in both enamel and dentin. Dentin close to the DEJ showed an average hardness number 62%-69% of control. Characterization of Mmp20-/- mouse dentin revealed a significant reduction in dentin thickness and mineral density and a transient increase in predentin thickness, indicating disturbances in dentin matrix secretion and mineralization. CONCLUSION: These results expand the spectrum of MMP20 disease-causing mutations and provide the first evidence for MMP20 function during dentin formation.
Assuntos
Amelogênese Imperfeita/genética , Metaloproteinase 20 da Matriz/genética , Mutação , Alelos , Amelogênese Imperfeita/patologia , Animais , Esmalte Dentário/patologia , Dentina/patologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , LinhagemRESUMO
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.
Assuntos
Proteômica/métodos , Animais , Cromatografia Líquida , Cemento Dentário/metabolismo , Dentina/metabolismo , Matriz Extracelular/metabolismo , Camundongos , Microdissecção , Odontogênese/genética , Odontogênese/fisiologia , Análise de Componente Principal , Proteoma/análise , Espectrometria de Massas em TandemRESUMO
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.