Immunohistochemical staining for identification of SLRPs.

<p>a) Light micrograph at 10X using immunohistochemical methods shows representative localization of SLRPs (biglycan expression in a 6 month old rat) counterstained with hematoxylin. Black arrows indicate localization at mesial PDL-bone enthesis spanning the root length as well as the coronal predominance. In addition localization was also observed in precementum layers (black arrow heads, in Fig. 5a) and at the CDJ (black arrow in inset, Fig. 5a), predentin, and the transseptal fibers (asterisk, Fig. 5a) between any two molars. Scale bars = 500 µm. b) Serially sectioned 60X light micrographs show immunohistochemical localization of SLRPs: biglycan (BGN), decorin (DCN), fibromodulin (FMOD), asporin (ASP), and negative controls (NC). Localization indicates more intense staining in coronal regions relative to apical regions across all age groups. All micrographs indicate regions where PDL meets primary bone (B) and primary cementum (PC) or secondary cementum (SC), with adjacent dentin (D). All Scale bars = 100 µm.</p

Histomorphometry as a function of age.

<p>a) Representative light micrograph of first molar distal root at 4X magnification illustrates histomorphometric parameters including i) CEJ-ABC distance, radial widths of: ii) primary cementum, iii) secondary cementum (SC), iv) PDL adjoining primary cementum (PDL-PC), and v) PDL adjoining secondary cementum (PDL-SC). Scale bar = 1 millimeter. b) The table illustrates increasing values of CEJ-ABC distance signifying recession with age. c) Plotted histomorphometric parameters as a function of age comparatively demonstrates increasing cementum and decreasing PDL trends with age.</p

Localization of sulfated GAGs using alcian blue stain.

<p>a) Light micrographs at 10X of alcian blue with nuclear fast red counterstain indicates more sulfated GAGs on the mesial aspect as indicated by black arrows (shown here in a 1.5 month old rat), as well as around osteocytes in the alveolar bone, precementum (white arrows) and cementum dentin junction (white asterisks). We also observed staining to occur in dentin. Scale bars = 500 µm. b) Corresponding age-related changes at interradicular, coronal-apical, and mesial-distal entheses regions showed decreased alcian blue staining and lost mesial specificity with age (black arrows). Scale bars = 100 µm.</p

Elemental mapping using BSE and EDS.

<p>a) SEM in BSE mode indicated distribution of high and low Z elements of the periodontium. Scale Bar = 0.5 mm. b) Using SEM-BSE greatest heterogeneity is observed in the different layers of bone owing to different chemical compositions of bone during its turnover (i.e. remodeling or modeling). Scale Bar = 100 µm. Representative point shoot EDS demonstrate varying counts of Ca and P (Kα1, Kα2, and Kβ1 lines) within adjacent regions. SE mode indicates at length scales c) the enthesis region of PDL-bone with scale bar = 100 µm, d) the site of a resorption pit, which indicate the digested layers of bone with scale bar = 5 µm, and e) the exposed collagen fibers that help compose a mineralized matrix with scale bar = 4 µm.</p

Morphological changes and anatomical locations within TRAP positive cells.

<p>Morphological changes were observed in histological sections and confirmed in three dimensional space using a MicroXCT (shown in insets comparing occlusal, distal, and mesial surfaces). The distal preference of TRAP positive cells and progressive mesial curving were observed. Anatomical structures include enamel space (ES), dentin (D), periodontal ligament (PDL), alveolar bone (AB), and secondary cementum (SC). TRAP stained micrographs at 4X magnification show first molar distal roots decreasing in osteoclast intensity and frequency with age. Representative coronal and apical TRAP positive areas in the encircled regions are enlarged in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035980#pone-0035980-g003" target="_blank">Figure 3</a>. With age, a transition from CEJ to DGJ (demarcated by white asterisk) and increasing recession (CEJ-ABC distance represented by distance between dashed red lines) were observed. Scale bar = 1 millimeter. CEJ: cementoenamel junction; DGJ: dentin gingival junction; ABC: alveolar bone crest.</p