Proteomic analysis of human dental cementum and alveolar bone

Abstract

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.

Keywords: Alveolar bone; Dental cementum; Dentin; Periodontal ligament; Proteomic analysis; Superoxide dismutase 3.

Figure 1. Proteomic profiles of human dental cementum and alveolar bone

(A) Diagram of tooth indicating dental cementum (DC) and alveolar bone (AB), as well as periodontal ligament (PDL), dentin (DENT), and enamel (ENAM). (B) Venn diagram of proteins identified by LC-MS/MS exclusive to alveolar bone (105) versus dental cementum (83), and those found in both tissues (130). Predicted (C) cellular distributions and (D) molecular function of proteins identified in human alveolar bone and dental cementum, generated by DAVID software using GOTERM_CC_3 and GOTERM_MF_ALL databases. Gene Ontology (GO) terms are represented as different color wedges in the pie charts, with the number of proteins per group shown in parentheses.

Figure 2. Localization of dental cementum and alveolar bone enriched proteins in mouse tissues

Immunohistochemistry was used to confirm presence of selected proteins enriched in cementum (A–J) or bone (K–L) based on proteomics analysis. (A, B) Osteopontin (OPN) is noted in the dental cementum (DC) matrix and alveolar bone (AB), especially at reversal lines. (C, D) Biglycan is richly present in the periodontal ligament (PDL), and also localized to cementocytes (Ccy; inset) of the DC. (E, F) Periostin (POSTN) localizes heavily to PDL, and Sharpey’s fibers (SF; inset) entering into DC are observed to be positive for POSTN. (G, H) Fibromodulin (FMOD) is found in high concentrations in the PDL matrix, and also produced by cementocytes (inset). (I, J) Superoxide dismutase 3 (SOD3) is found in PDL and cementum matrix, with cementocytes staining positive (inset). (K, L) Decorin (DCN) is found in PDL matrix and Sharpey’s fibers (SF; inset) entering alveolar bone. (M, N) Negative control slides display lack of staining in the absence of primary antibody. All images are from 60 dpn mouse mandibular molar teeth and surrounding tissues. Yellow dotted lines are used to mark the cementum-dentin interface.

Figure 3. SOD3 localization in cementum and associated cells

In a mouse mandible at 26 dpn, SOD3 localizes most strongly to dental cementum (DC) of the (A) first molar, (B) second molar, and (C) incisor teeth (black arrows), with slight staining in the surrounding alveolar bone (AB). Immunohistochemistry in (D) mouse femur at 30 dpn identifies (E) light SOD3 in bone of the metaphysis (MET) and epiphysis (EPI), though not in growth plate (GP) cartilage, with little or no SOD3 found in (F) trabecular bone (TB) or cortical bone (CB) distal to the growth plate. (G) Q-PCR on 15 dpn mouse tissues reveals significantly higher SOD3 mRNA in molar teeth compared to femurs, calvariae, or brain tissues (p < 0.05 by ANOVA, where a different capital letter indicates significant intergroup difference). In human teeth, strong SOD3 staining is found in (H) cementoblasts (Cb) and the periodontal ligament (PDL) closest to the acellular form of cementum, as well as in (I) cementocytes (Ccy) of the cellular DC. (J) SOD3 is also observed in odontoblasts (Od) and their processes extending into the dentin matrix (DENT) in human teeth. (K) Similar to immunolocalization in human teeth, pig molars displayed strongest SOD3 localization in cementoblasts and cementocytes of the cellular DC, as well as cells in the AB and PDL.