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, 50 (6), 870-80

Differentiating Zones at Periodontal Ligament-Bone and Periodontal Ligament-Cementum Entheses

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Differentiating Zones at Periodontal Ligament-Bone and Periodontal Ligament-Cementum Entheses

J-H Lee et al. J Periodontal Res.

Abstract

Background and objective: The structural and functional integrity of bone-periodontal ligament (PDL)-cementum complex stems from the load-bearing attachment sites (entheses) between soft (PDL) and hard (bone, cementum) tissues. These attachment sites are responsible for the maintenance of a bone-PDL-cementum complex biomechanical function. The objective was to investigate changes in spatiotemporal expression of key biomolecules in developing and functionally active entheses.

Material and methods: Multilabeling technique was performed on hemimandibles of 3 wk and 3 mo-old scleraxis-GFP transgenic mice for CD146, CD31, NG2, osterix and bone sialoprotein. Regions of dominant stretch within the PDL were evaluated by identifying directionality of collagen fibrils, PDL fibroblasts and PDL cell cytoskeleton.

Results: CD146+ cells adjacent to CD31+ vasculature were identified at PDL-bone enthesis. NG2+ cells were located at coronal bone-PDL and apical cementum-PDL entheses in the 3-wk-old group, but at 3 mo, NG2 was positive at the entheses of the apical region and alveolar crest. NG2 and osterix were colocalized at the osteoid and cementoid regions of the PDL-bone and PDL-cementum entheses. Bone sialoprotein was prominent at the apical region of 3-wk-old mice. The directionality of collagen fibers, fibroblasts and their cytoskeleton overlapped, except in the apical region of 3 wk.

Conclusion: Colocalization of biomolecules at zones of the PDL adjacent to attachment sites may be essential for the formation of precementum and osteoid interfaces at a load-bearing bone-PDL-tooth fibrous joint. Biophysical cues resulting from development and function can regulate recruitment and differentiation of stem cells potentially from a vascular origin toward osteo- and cemento-blastic lineages at the PDL-bone and PDL-cementum entheses. Investigating the coupled effect of biophysical and biochemical stimuli leading to cell differentiation at the functional attachment sites is critical for developing regeneration strategies to enable functional reconstruction of the periodontal complex.

Keywords: periodontal ligament; tissue entheses; tooth-bone biomechanics.

Figures

Figure 1
Figure 1. Co-immunofluorescent staining of CD146 with endothelial cell marker, CD31 in scxGFP mouse 2nd molar at 3 weeks (A-E) and 3 months (F-J) of age
(A, F) CD146+ cells were detected in association with vasculature identified by CD31, through subginigival connective tissue, PDL space, dental pulp, endosteal space, and bone marrow. (B-E, G-J) Magnified view of the inserted insets in panel A and F. CD146+ cells are derived from endosteal spaces through the channels between alveolar bone and PDL (arrowhead). Scale bar; 200μm (A,F), 100μm (B-E, G-J)
Figure 2
Figure 2. The distribution of CD 146+, CD31+ Cells in the vicinity of bone-PDL interface
(A) The fluorescent intensity of CD146 and CD31 was measured within 25μm (a), 50μm (b), and 100μm (c) from B to PDL interface of mesial complex of mandibular 2nd molar, Scale bar=100μm. (B) Both CD31 and CD146 showed significantly higher fluorescence within 25μm and 50μm compared to 100μm at both ages * statistically significance (p<0.05), N=5, error bars present the standard error of the mean (SEM)
Figure 3
Figure 3. Co-immunofluorescent staining of NG2+ cells with endothelial cell marker, CD31 in scxGFP mouse 2nd molar at 3 weeks (A-C) and 3 months (D-F) of age
(A,D at X20) NG2+ cells (red) were observed in proximity of CD31+ blood vessels (yellow) but also at bone-PDL and cementum-PDL interfaces. (B, E at X60) Magnified view of interradicular region. Interradicular region was intensely populated with CD31+ blood vessels (*) closely to NG2+ cell-lining bone-PDL interface. (C, F at X60) PDL adjacent to bone. NG2+ cells are located adjacent to CD31+ endothelial wall (*) within PDL. b=bone, d=dentin, Scale bar; 200μm (A, D), 20μm (B,C,E and F)
Figure 4
Figure 4. Immunofluorescent localization of NG2+ (A-H) and Osx+ (I-P) cells in scxGFP mouse 2nd molar at 3 weeks (A-D and I-L) and 3 months (E-H and M-P) of age
(A-H) NG+ cells (red) were observed along bone-PDL and cementum-PDL interfaces. Bone-PDL interface had NG2+ cells coronally (B, D) but not apically (C, arrowheads), although 3 months-old mice showed increased NG expression in apical region (G) as well as on alveolar crest (F) and interradicular bone (H). On the other hand, NG+ cells at cementum-PDL interface of 3 weeks-old mice, were concentrated in the region where cellular cementum is actively developing (C). This pattern was also observed in 3 months-old mice but not as obvious as in 3 weeks-old mice. (I-P) Osx+ cells (red or pink) were detected at the location of NG2+ cells along interface: bone-PDL attachment site (J,L,N and P) except apical bone-PDL interface (K, arrowheads) and cementum-PDL interface (K, O). Moreover, Osx was positive in odontoblasts lining pulp chamber and root canal (I and M), bone marrow stromal cells (I,M and O) and some Scx+ PDL fibroblasts. b=bone, d=dentin, c=cementum, bm=bone marrow, Scale bar=200um (A,E,I and M), 50um (B-D, F-H, J-L, and N-P)
Figure 5
Figure 5. Immunofluorescence of BSP in scxGFP mouse 2nd molar at 3 weeks (A-C) and 3 months (D-F) of age
BSP (red) is localized to alveolar bone (b), acellular cementum (ac) and cellular cementum (cc) at both ages. BSP was evenly distributed in coronal region (A,D) and interradicular bone (C,F) with a rich expression in cement lines of apical bone (B,E), However, BSP is enriched at the apical bone-PDL interface at 3 weeks (B, arrowheads), whereas the apical bone-PDL interface at 3 months is BSP negative (E, arrowheads). b= alveolar bone, d=dentin, ac=acellular cementum, cc =cellular cementum, Scale bar = 50μm
Figure 6
Figure 6. Orientation and organization of PDL cell cytoskeleton, fibroblasts and collagen fibers
Immunofluorescence of phalloidin (red) and polarized light microscopy of PSR staining in scxGFP 2nd molar at 3 weeks (A) and 3 months (B). Plots illustrate directionality of birefringent collagen fibers, fibroblasts, and PDL cell cytoskeletons within mesiocoronal (i,iii) and mesioapical regions (ii,iv). Directionality of collagen fibers, fibroblasts, and PDL cell cytoskeleton coincides within PDL complex, except in apical region of 3 weeks old group. b=alveolar bone, d=dentin, cc=cellular cementum, Scale bar = 100μm
Figure 7
Figure 7. Schematic representation of the distribution of biomolecules identified in scxGFP mice molar at 3 weeks and 3 months
The alveolar bone crest and periapical region are areas of high biomechanical activity and the entheses within those regions are putative locations of cellular differentiation and tissue adaptation. Perivascular niche for stemness identified using CD146 and CD31 (yellow) runs along bone-PDL entheses. NG2/Osx+ cells line the bone-PDL and cementum-PDL entheses, where biophysical signals from eruption and function are supposedly amplified. Scx are positive only in the fully developed PDL as a conveyer of biophysical cues. BSP was rich at apical bone-PDL enthesis at 3 weeks but negative at 3 months

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