Global proteome profiling of dental cementum under experimentally-induced apposition

Abstract

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.

Keywords: Dental cementum; Periodontal ligament; Proteomic analysis; Tissue apposition.

Figure 1. Experimentally-induced dental cementum apposition model in mouse

(A) Extraction of first (M1) and second (M2) maxillary molars. The antagonist lower molars were kept out of occlusion, inducing super-eruption and apposition of dental cementum (DC) and alveolar bone (AB). (B) Light and (C) fluorescence microscopy images of a longitudinal section of the mesial root of the first mandibular molar. Blue dotted lines in B indicate the DC-dentin (D) border. The white arrows in panel C point to the fluorescent labels formed by the incorporation of fluorochrome markers during DC apposition. Calcein (administered at 24 hours and 17 days after surgery) is observed as two intense green lines, while tetracycline (administered 9 days after surgery) is observed as the faint yellow line between the tetracycline labels.

Figure 2. Proteomic profile of dental cementum

Area-proportional Venn diagram showing the distribution of the total 519 proteins identified in dental cementum (DC) under experimentally-induced apposition for 6 and 21 days (EIA groups), as well as respective control groups at the same time points. Proteins were considered as an identified protein when detected in, at least, one of the seven individual samples. The number of exclusive proteins to each group and shared proteins in every intersection of the diagram are shown.

Figure 3. Immunohistochemistry for identification of dental cementum markers

Immunohistochemistry (IHC) was used to localize (by red or brown-red reaction color) selected protein markers identified by proteomic analysis of dental cementum (DC). All photomicrographs were taken at original magnification of 100×. Blue dotted lines in all panels indicate the DC-dentin (DE) border. (A) Collagen type I alpha 1 (COL1A1) localizes to DC, DE, and alveolar bone (AB). (B) Collagen type XII (COL12) localizes to periodontal ligament (PDL) and DC-PDL interface. (C) Bone sialoprotein (BSP) stains DC and AB. (D) Osteopontin (OPN) is found in DC and AB. (E) Dentin matrix protein 1 (DMP1) is found in AB and DC, around osteocytes and cementocytes, respectively, as well as in tubules of DE. (F) Tissue nonspecific alkaline phosphatase (TNAP) is rich in the PDL and at the borders of DC-PDL and AB-PDL. (G) Decorin (DCN) localizes to PDL and DC-PDL interface. (H) Biglycan (BGN) is found in DC and PDL. (I) Fibromodulin (FMOD) is observed in PDL, and in DC around cementocytes lacunae. (J) Asporin (ASPN) stains the PDL and DC-PDL interface. (K) Periostin (POSTN) is richly present in the PDL and associated with embedded collagen fibers in DC and AB. (L) A negative control (NEG) section that lacked primary antibody is included for comparison to IHC-stained sections.

Figure 4. Dental cementum protein expression is significantly regulated by experimentally-induced apposition

(A) Venn diagram showing the distribution of 24 differentially expressed proteins under the effect of EIA alone, or including the interaction with time. Identities and fold-changes for these 24 statistically significant differentially expressed proteins are described in bar graphs in panels B–D. (B) Bar graph showing the fold-regulation for 12 proteins under the exclusive effect of EIA, at 6 and 21 days. (C) Bar graph showing the fold-regulation of 6 proteins at 6 days. (D) Bar graph showing the fold-regulation of 6 proteins at 21 days.

Figure 5. Immunohistochemistry confirmation of proteins in control and experimentally-induced apposition tissues

Immunohistochemistry (IHC) was used to localize (by red or brown-red reaction color) selected protein markers identified by proteomic analysis of dental cementum (DC) in control and experimentally-induced apposition (EIA) groups 21 days after surgery. All photomicrographs were taken at original magnification of 100×. Blue dotted lines in all panels indicate the DC-dentin (DE) border. (A, B) Asporin (ASPN) localizes to alveolar bone (AB), periodontal ligament (PDL) and DC in control and EIA tissues. (C, D) Collagen type XI alpha 1 (COL11A1) exhibits immunostaining in AB, PDL, DE, and DC in control and EIA tissues. (E, F) Secreted protein, acidic cysteine-rich (SPARC) immunostains widely in AB, PDL, DE, and DC in control and EIA groups. (G, H) Osteopontin (OPN) localizes to reversal lines in AB, as well as DC matrix in control and EIA tissues. (I, J) Fibromodulin (FMOD) is observed immunostaining AB, PDL, DE-pulp, and DC in control and EIA tissues.

Figure 6. Temporal changes in the dental cementum proteomic profile

Bar graph showing the distribution of selected differentially expressed proteins over time in dental cementum (DC). (*) proteins regulated over time in the experimentally-induced apposition (EIA) groups.

Figure 7. Enriched gene ontology groups in dental cementum under experimentally induced apposition

Enriched gene ontology (GO) terms of 56 differentially expressed proteins in dental cementum (DC) after experimentally-induced apposition (EIA) at 6 and 21 days, generated by the Functional Classification tool using GO Molecular function category in PANTHER Classification System. Only statistically significantly enriched GO terms are displayed, with the percentage of genes that hit compared against the total number of hits.

Figure 8. Gene ontology evidence of cementocyte activation by experimentally induced apposition

Bar graphs indicating (A) GO-Slim cellular component and (B) molecular function classification of each protein group list were compared to the Mus musculus reference database using the binomial test, followed by Bonferroni correction, on the Statistical Overrepresentation Test in the PANTHER Classification System for gene ontology analysis. PANTHER-generated graphics display only significantly enriched terms. Blue and yellow bars represent significant terms with p-values <0.05 and <0.01, respectively. Arrows indicates child categories of molecular function of selected enriched terms with corresponding protein list involved with the GO term. Differentially regulated proteins for each term are listed in the boxes.

Figure 8. Gene ontology evidence of cementocyte activation by experimentally induced apposition

Bar graphs indicating (A) GO-Slim cellular component and (B) molecular function classification of each protein group list were compared to the Mus musculus reference database using the binomial test, followed by Bonferroni correction, on the Statistical Overrepresentation Test in the PANTHER Classification System for gene ontology analysis. PANTHER-generated graphics display only significantly enriched terms. Blue and yellow bars represent significant terms with p-values <0.05 and <0.01, respectively. Arrows indicates child categories of molecular function of selected enriched terms with corresponding protein list involved with the GO term. Differentially regulated proteins for each term are listed in the boxes.