Methods for studying tooth root cementum by light microscopy

Abstract

The tooth root cementum is a thin, mineralized tissue covering the root dentin that is present primarily as acellular cementum on the cervical root and cellular cementum covering the apical root. While cementum shares many properties in common with bone and dentin, it is a unique mineralized tissue and acellular cementum is critical for attachment of the tooth to the surrounding periodontal ligament (PDL). Resources for methodologies for hard tissues often overlook cementum and approaches that may be of value for studying this tissue. To address this issue, this report offers detailed methodology, as well as comparisons of several histological and immunohistochemical stains available for imaging the cementum-PDL complex by light microscopy. Notably, the infrequently used Alcian blue stain with nuclear fast red counterstain provided utility in imaging cementum in mouse, porcine and human teeth. While no truly unique extracellular matrix markers have been identified to differentiate cementum from the other hard tissues, immunohistochemistry for detection of bone sialoprotein (BSP), osteopontin (OPN), and dentin matrix protein 1 (DMP1) is a reliable approach for studying both acellular and cellular cementum and providing insight into developmental biology of these tissues. Histological and immunohistochemical approaches provide insight on developmental biology of cementum.

Figure 1

Schematic of the mouse molar tooth. The illustrated image is modeled after a coronal buccolingual section of a mouse first mandibular molar, and the mineralized tissues of the dentoalveolar are emphasized by color enhancement: enamel (blue), dentin (yellow), cementum (green for both acellular and cellular varieties) and alveolar bone (orange). The pulp chamber, PDL and gingiva are also labeled as relevant soft tissues. AEFC, acellular extrinsic fiber cementum; CIFC, cellular intrinsic fiber cementum; PDL, periodontal ligament.

Figure 2

Histological stains for cementum. Serial sections of the 44 dpn mouse mandibular first molar were stained with (a–d) H&E, (e–h) TB and (i–l) AB-NFR. Visualization of AEFC and CIFC is emphasized in the accompanying text. A dotted line is used to highlight the circumference of the CIFC in panels (b), (f) and (j). Original magnifications (×50, ×100, ×200 and ×400) are labeled over the panels. AB-NFR, Alcian blue and nuclear fast red; AEFC, acellular extrinsic fiber cementum; CIFC, cellular intrinsic fiber cementum; dpn, days postnatal; H&E, hematoxylin and eosin; TB, toluidine blue.

Figure 3

PR staining of the cementum–periodontal ligament complex. Mouse first molar sections at 44 dpn stained by the PR method were viewed under (a–d) polarized light to visualize AEFC and CIFC and collagen organization and insertion from the PDL. The same sections under (e–h) non-polarized light are displayed to indicate overall tissue morphology. A dotted line is used to highlight the circumference of the CIFC in panels (b) and (f). Original magnifications (×50, ×100, ×200 and ×400) are labeled over the panels. AEFC, acellular extrinsic fiber cementum; CIFC, cellular intrinsic fiber cementum; dpn, days postnatal; PDL, periodontal ligament; PR, Picrosirius red.

Figure 4

Staining to detect cementum-periodontal ligament defects in the alkaline phosphatase deficient mouse. WT and alkaline phosphatase null (Alpl^−/−) first mandibular molar tissues from 14 dpn mice were compared by AB-NFR and PR staining. AB-NFR staining reveals a layer of nascent acellular cementum (AEFC) in (a and b) WT molar, while this layer was confirmed to be absent in the age-matched (c and d) Alpl^−/− molar. PR staining under polarized light indicated that (e and f) collagen fibers were becoming organized in the WT molar PDL at this age; however, (g and h) Alpl^−/− molar PDL was severely disorganized with signs of periodontal detachment (*) from the molar surface. Dotted boxes in panels (a), (c), (e) and (g) represent regions shown at higher magnification in panels (b), (d), (f) and (h), respectively. Original magnification was ×100 for (a), (c), (e) and (g), and ×400 for (b), (d), (f) and (h). AB-NFR, Alcian blue and nuclear fast red; AEFC, acellular extrinsic fiber cementum; dpn, days postnatal; PDL, periodontal ligament; PR, Picrosirius red; WT, wild-type.

Figure 5

Histological staining of porcine and human cementum. Sections of the first mandibular molar of a 13- to 16-week-old Hanford miniature pig and human third molar were used to contrast H&E and AB-NFR staining for visualizing cementum (CEM). (a and b) H&E and (e and f) AB-NFR staining of cellular cementum of the porcine cervical root provides contrast for observation of the CDJ (arrowheads in panels a and e) and cementum/PDL interface, as well as embedded cementocytes (cyte in panels b and f). In human molar, staining was evaluated for (c and d) mixed stratified cementum of the furcation area and (g and h) acellular cementum of the cervical root. Both H&E and AB-NFR provide contrast for the initial cementum layer in mixed stratified (c and g) and acellular cementum (d and h), while AB-NFR provides more contrast between acellular cementum and cells of the PDL. Original magnifications (×100 and ×400) are labeled over the panels. AB-NFR, Alcian blue and nuclear fast red; CDJ, cementum–dentin junction; H&E, hematoxylin and eosin; PDL, periodontal ligament.

Figure 6

Immunohistochemistry for cementum. Mouse first mandibular molar sections at 44–45 dpn were immunostained for (a–c) BSP, (d–f) OPN and (G–I) DMP1. Both BSP and OPN localize to acellular and cellular cementum (AEFC and CIFC, respectively), as well as bone, though OPN is more widely distributed, such as in PDL. DMP1 localizes to the cementum–dentin junction region, as well as cellular cementum of the apex. A dotted line is used to highlight the circumference of the CIFC in panels (b) and (h). Original magnifications (×50, ×100 and ×200) are labeled over the panels. AEFC, acellular extrinsic fiber cementum; BSP, bone sialoprotein; CIFC, cellular intrinsic fiber cementum; DMP1, dentin matrix protein 1; dpn, days postnatal; OPN, osteopontin; PDL, periodontal ligament.