The Development of Dentin Microstructure Is Controlled by the Type of Adjacent Epithelium

Abstract

Considerable amount of research has been focused on dentin mineralization, odontoblast differentiation, and their application in dental tissue engineering. However, very little is known about the differential role of functionally and spatially distinct types of dental epithelium during odontoblast development. Here we show morphological and functional differences in dentin located in the crown and roots of mouse molar and analogous parts of continuously growing incisors. Using a reporter (DSPP-cerulean/DMP1-cherry) mouse strain and mice with ectopic enamel (Spry2^+/- ;Spry4^-/- ), we show that the different microstructure of dentin is initiated in the very beginning of dentin matrix production and is maintained throughout the whole duration of dentin growth. This phenomenon is regulated by the different inductive role of the adjacent epithelium. Thus, based on the type of interacting epithelium, we introduce more generalized terms for two distinct types of dentins: cementum versus enamel-facing dentin. In the odontoblasts, which produce enamel-facing dentin, we identified uniquely expressed genes (Dkk1, Wisp1, and Sall1) that were either absent or downregulated in odontoblasts, which form cementum-facing dentin. This suggests the potential role of Wnt signalling on the dentin structure patterning. Finally, we show the distribution of calcium and magnesium composition in the two developmentally different types of dentins by utilizing spatial element composition analysis (LIBS). Therefore, variations in dentin inner structure and element composition are the outcome of different developmental history initiated from the very beginning of tooth development. Taken together, our results elucidate the different effects of dental epithelium, during crown and root formation on adjacent odontoblasts and the possible role of Wnt signalling which together results in formation of dentin of different quality. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Keywords: DENTIN; DENTINOGENESIS; INCISOR; LIBS; MICROSTRUCTURE; MOLAR; ODONTOBLAST; ODONTOGENESIS; PROCESSES; TEETH; WNT SIGNALING.

Fig 1

Dentin microstructure in molar and incisor: root (analogue) versus crown (analogue). Confocal images from thick, cleared histological sections from the DSPP ^Cerulean /DMP1 ^Cherry mouse reporter strain show details of the microstructure of dentin with the focus on odontoblasts' processes in different parts of teeth. The sagittal section of the first molar (A) and transversal section of the mandibular incisor (D) show the structural similarities between these two types of teeth and the distribution of fluorescent proteins in the dental tissue and its close surroundings. Note the expression of cherry (DMP1) in odontoblasts but also bone‐forming osteoblasts(cytes) and cementum‐forming cementoblasts(cytes) on the surface of roots, while cerulean (DSPP) is expressed specifically by odontoblasts. Subsets of A and B show details of enamel‐facing (crown/crown‐analogue) dentin (B, E) and cementum‐facing (root/root analogue) dentin (C, F), respectively. Arrowheads highlight the presence of DMP1‐expressing cementum‐forming cells located around the root of molar and root analogue of incisors. Schematic visualization of the microstructure of dentin showing the same pattern in enamel‐facing and cementum‐facing aspects in both molars and incisors (G). Scale bars = 300 μm (A, D), 100 μm (B, C, E, F). Ab = alveolar bone; De = dentin; en = enamel; Ob = odontoblasts; P = pulp.

Fig 2

Mandibular and maxillary incisor dentin microstructure. Both upper and lower continuously growing incisors (A, B) show the same pattern in the labial (enamel‐facing) (C, E) and lingual (cementum‐facing) (D, F) odontoblast processes, respectively. White asterisks mark different microstructure of odontoblast processes in the lingual (cementum‐facing) aspect of both mandibular and maxillary incisors. Scale bars = 1000 μm (A, B) and 50 μm (C–F) for details. En = enamel; PDL = periodontal ligaments.

Fig 3

Dentin adjacent to ectopically developed enamel shows altered microstructure. Confocal images of the labial and lingual aspect of maxillary incisors stained with Phalloidin‐Alexa488 show the different microstructure of OPs on the labial and lingual aspect of incisor in wild‐type animals (A) and similar microstructure on both aspects in maxillary incisor of Spry2 ^+/− ;Spry4 ^−/− animals (B) having ameloblasts and enamel on both sides of the tooth. (C) Schematic drawing reflecting the influence of different dental epithelium on dentin development. Red asterisk shows the altered morphology of OPs in dentin adjacent to ectopic enamel on the lingual aspect of the maxillary incisor in Spry2^+/−;Spry4^−/− . Scale bars = 50 μm. En = enamel space; LaCL = labial cervical loop; LiCL = lingual cervical loop; PDL = periodontal ligament.

Fig 4

Analysis of odontoblasts' terminal branching regions and processes straightness in enamel‐facing and cementum‐facing dentin. Confocal images of histological sections from DSPP ^Cerulean /DMP1 ^Cherry , wild‐type, and Spry2 ^+/− ;Spry4 ^−/− animals stained with Phalloidin‐Alexa488 with a special emphasis on the terminal branching regions relating to the first formed dentin adjacent to enamel and cementum, respectively (A). Details on the process morphology are shown in (B) and quantifications straightness of the processes in (C). Red asterisks show the altered morphology of OPs in dentin adjacent to ectopic enamel on the lingual aspect of the maxillary incisor in Spry2 ^+/− ;Spry4 ^−/− . Scale bars = 20 μm. Cr = crown (analogue); en = enamel/enamel space; Lab. = labial side; Ling. = lingual side; PDL = periodontal ligament; Ro = root (analogue).

Fig 5

Characterization of the features of processes and dentin. Quantification of different aspects of odontoblast processes and dentin quality in mandibular wild‐type and maxillary wild‐type and Spry2 ^+/− ;Spry4 ^−/− animals. Process width (A) measured as a width of individual odontoblast processes at the distance of 40 μm from the predentin‐pulp edge (n = 85). Process density (B) calculated as a number of OPs per 100 μm² of dentin (n = 4 ROIs). Fraction of dentin height without the presence of the main process (C) (n = 33). Analysis of the distribution of all processes in dentin ranging from predentin (1) to the outermost dentin (10) is shown in (D). Pearson's correlation coefficients (R) were calculated and tested. p < 0.05 expresses the fact that trends are correlated (n = 3 ROIs for each data point). Ect. enamel = ectopic enamel; Lab. = labial side; Ling. = lingual side.

Fig 6

Early polarization of odontoblast and dentin patterning. Confocal images of histological sections from maxillary incisors of wild‐type and Spry2 ^+/− ;Spry4 ^−/− animals stained with Phalloidin‐Alexa488 (green) and DAPI (blue). Each panel contains an overview image of the early differentiating area where preodontoblasts polarize and differentiate. Highlighted subsets show details of differentiation. In the wild‐type mouse, epithelium at the labial aspect gives rise to (pre)ameloblasts (highlighted with white arrowheads A), whereas at the lingual aspect, preameloblasts are not formed and the epithelium is being disintegrated (B). In contrast to this, the enamel‐forming ameloblast epithelium is formed by both labial and lingual cervical loops of Spry2 ^+/− ;Spry4 ^−/− incisors. Ectopic, (pre)ameloblastic epithelium is marked with red arrowheads. Asterisks highlight the difference between the structure of wild‐type and Spry2 ^+/− ;Spry4 ^−/− lingual cervical loop highlighting different interacting partners of the respective (pre)odontoblasts. Note faster polarization of odontoblasts induced by adjacent epithelium with ameloblasts at the labial aspect of wild‐type and both aspects of Spry2 ^+/− ;Spry4 ^−/− incisors (A, C, D). Scale bars = 100 μm (cervical loop tile scans), 25 μm (details). Ab = ameloblasts; pAb = preameloblasts; De = dentin; En = enamel space; LaCL = labial cervical loop; LiCL = lingual cervical loop; Ob = odontoblasts; pOb = preodontoblasts; P = pulp; PDL = periodontal ligament.

Fig 7

Reporter expression distance analysis. Analysis of images taken from DSPP ^Cerulean /DMP1 ^Cherry mandibular (A) and maxillary (C) incisor's apical regions. The distance from the apex of the cervical loop to the first cell expressing fluorescent protein: DSSP (cerulean), DMP1 (cherry), or both were measured and results were compared between the lingual and labial aspects (B, D). Scale bars = 200 μm. Arrows indicate cementoblasts. Ab = alveolar bone; LaCL = labial cervical loop; LiCL = lingual cervical loop; PDL = periodontal ligament.

Fig 8

Molecular differences of odontoblasts on the labial and lingual aspect of mouse incisor. Analysis of selected odontoblast‐specific molecular markers shows a different expression pattern on the labial and lingual aspect of both mandibular and maxillary incisors. In situ hybridization of Dkk1 (A, B) and Wisp1 (C, D) shows a specific expression only in odontoblasts on the labial aspect of the incisor. Similar to this, the immunohistochemical staining of SALL1 shows the same expression pattern (E, F), while the immunohistochemical staining of previously shown NFIC (G, H) did not show a different expression on labial and lingual aspects. Scale bars = 200 μm (overview images) and 50 μm (details). LaCL = labial cervical loop; LiCL = lingual cervical loop; Ab = ameloblasts; pAb = preameloblasts; Ob = odontoblasts; pOb = preodontoblasts; PDL = periodontal ligament.

Fig 9

Analysis of elemental composition and dentin density. Brightfield images (left) of ground sectioned, polished teeth sections and subsequent analyses (right) represent heat maps showing the relative amount of calcium and magnesium on the transversal section from the distal part of the mandibular and maxillary incisor (A). The function of position plots of principal component analysis (B) and k‐means clustering from the PCA (D). Micro‐CT analysis of dentin density in EFD and CFD of mandibular and maxillary incisors (C) (n = 25). Graphs also plot the grey mean values of enamel to enable the comparison between dentin and enamel. Scale bars = 500 μm. AB = alveolar bone; CFD = cementum‐facing dentin; EFD = enamel‐facing dentin; En = enamel; PDL = periodontal ligament.