The periodontal ligament (PDL) is a unique connective tissue mainly comprising collagen fiber bundles and cells between the roots of teeth and inner walls of the alveolar-bone socket. PDL fiber bundles are arrayed between teeth and bone, with both ends embedded in the cementum or alveolar bone as Sharpey's fiber. These bundles, synthesized by PDL fibroblasts (PDLFs), form several distinct groups within the PDL which has important functions besides tooth anchoring including tooth nutrition, proprioception, sensory detection, homoeostasis, and repair of damaged tissue. However, little is known about how the regular-PDL fiber bundle arrays are formed, maintained, and remodeled over large distances from cementum to alveolar bone. Recently, novel instruments and 3D-imaging methods have been developed that have been applied to the investigation of hard tissues including the PDL. Work from our laboratory has revealed the three-dimensional (3D) ultrastructure of PDLFs and PDL collagen bundles by focused ion beam/scanning electron microscope tomography. We have shown that PDLFs have a flat shape with long processes or a wing-like shape, while PDL bundles are a multiple-branched structure wrapped in thin sheets of PDLF cytoplasm. Furthermore, PDLFs form an extensive cellular network between the cementum and alveolar bone. The PDL cellular network is presumed to synchronize PDL fiber bundles and regulate arrays of PDL fiber bundles via gap junctions. In this review, we summarize and discuss our current 3D-histomorphometric studies of the PDL at the mesoscale level.

This study was designed to investigate the role of the Wnt/β-catenin signaling pathway in estrogen-enhanced osteogenic differentiation of human peridontal ligament stem cells (hPLSCs). The limiting dilution technique was used for cloning and purification of hPLSCs. Flow cytometric analysis of STRO-1, CD146, and CD45 was conducted to identify hPLSCs. The P3 hPDLSCs were divided into 4 groups: Control, 10M E2, 10M E2+100 ng/mL Wnt3a, 10M E2+5 × 10M Xav939. After 7 days of osteogenic induction, qRT-PCR was used to detect the mRNA expression of β-catenin, CyclinD1, alkaline phosphatase, Runx2, and OCN; Western blot was used to detect the protein expression of β-catenin, GSK3β, P-GSK3β, CyclinD1, Runx2, and OCN; After 1, 3, 5, 7 days of osteogenic induction, the activity of alkaline phosphatase was detected. The authors' results showed that E2 was able to enhance the osteogenic differentiation of hPDLSCs and Wnt/β-catenin signaling pathway was involved. Wnt3a activated the signaling pathway of Wnt/β-catenin and further enhanced the osteogenesis of hPDLSCs. Xav939 inhibited the Wnt/β-catenin signaling pathway in estrogen-mediated environment, but did not obviously inhibit the osteogenic differentiation of hPDLSCs. E2 enhanced osteogenic differentiation of hPDLSCs through the activation of the Wnt/β-catenin signaling pathway.

The cytokine receptor activator of NFκB ligand (RANKL) produced by osteocytes is essential for osteoclast formation in cancellous bone under physiological conditions, and RANKL production by B lymphocytes is required for the bone loss caused by estrogen deficiency. Here, we examined whether RANKL produced by osteocytes is also required for the bone loss caused by estrogen deficiency. Mice lacking RANKL in osteocytes were protected from the increase in osteoclast number and the bone loss caused by ovariectomy. Moreover, these mice did not exhibit the increase in bone marrow B lymphocytes caused by ovariectomy that occurred in control littermates. Deletion of estrogen receptor α from B cells did not alter B cell number or bone mass and did not alter the response to ovariectomy. In addition, lineage-tracing studies demonstrated that B cells do not act as osteoclast progenitors in estrogen-replete or estrogen-deficient mice. Taken together, these results demonstrate that RANKL expressed by osteocytes is required for the bone loss as well as the increase in B cell number caused by estrogen deficiency. Moreover, they suggest that estrogen control of B cell number is indirect via osteocytes and that the increase in bone marrow B cells may be a necessary component of the cascade of events that lead to cancellous bone loss during estrogen deficiency. However, the role of B cells is not to act as osteoclast progenitors but may be to act as osteoclast support cells. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Estrogen is critical for skeletal homeostasis and regulates bone remodeling, in part, by modulating the expression of receptor activator of NF-kappa B ligand (RANKL), an essential cytokine for bone resorption by osteoclasts. RANKL can be produced by a variety of hematopoietic (e.g. T and B-cell) and mesenchymal (osteoblast lineage, chondrocyte) cell types. The cellular mechanisms by which estrogen acts on bone are still a matter of controversy. By using murine reconstitution models that allow for selective deletion of estrogen receptor-alpha (ER alpha) or selective inhibition of RANKL in hematopoietic vs. mesenchymal cells, in conjunction with in situ expression profiling in bone cells, we identified bone lining cells as important gatekeepers of estrogen-controlled bone resorption. Our data indicate that the increase in bone resorption observed in states of estrogen deficiency in mice is mainly caused by lack of ER alpha-mediated suppression of RANKL expression in bone lining cells.

Specifics of the biochemical pathways that modulate collagen cross-links in the periodontal ligament (PDL) are not fully defined. Better knowledge of the collagen post-translational modifications that give PDL its distinct tissue properties is needed to understand the pathogenic mechanisms of human PDL destruction in periodontal disease. In this study, the post-translational phenotypes of human and mouse PDL type I collagen were surveyed using mass spectrometry. PDL is a highly specialized connective tissue that joins tooth cementum to alveolar bone. The main function of the PDL is to support the tooth within the alveolar bone while under occlusal load after tooth eruption. Almost half of the adult population in the USA has periodontal disease resulting from inflammatory destruction of the PDL, leading to tooth loss. Interestingly, PDL is unique from other ligamentous connective tissues as it has a high rate of turnover. Rapid turnover is believed to be an important characteristic for this specialized ligament to function within the oral-microbial environment. Like other ligaments, PDL is composed predominantly of type I collagen. Collagen synthesis is a complex process with multiple steps and numerous post-translational modifications including hydroxylation, glycosylation and cross-linking. The chemistry, placement and quantity of intermolecular cross-links are believed to be important regulators of tissue-specific structural and mechanical properties of collagens. Type I collagen was isolated from several mouse and human tissues, including PDL, and analyzed by mass spectrometry for post-translational variances. The collagen telopeptide cross-linking lysines of PDL were found to be partially hydroxylated in human and mouse, as well as in other types of ligament. However, the degree of hydroxylation and glycosylation at the helical Lys87 cross-linking residue varied across species and between ligaments. These data suggest that different types of ligament collagen, notably PDL, appear to have evolved distinctive lysine/hydroxylysine cross-linking variations. Another distinguishing feature of PDL collagen is that, unlike other ligaments, it lacks any of the known prolyl 3-hydroxylase 2-catalyzed 3-hydroxyproline site modifications that characterize tendon and ligament collagens. This gives PDL a novel modification profile, with hybrid features of both ligament and skin collagens. This distinctive post-translational phenotype may be relevant for understanding why some individuals are at risk of rapid PDL destruction in periodontal disease and warrants further investigation. In addition, developing a murine model for studying PDL collagen may be useful for exploring potential clinical strategies for promoting PDL regeneration. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Recent evidence demonstrated an interplay between estrogens and growth hormone (GH) at cellular level. To investigate the possible mechanism/s involved, we studied the effect of 17β-estradiol (E2) on GH signaling pathways in primary culture of human osteoblasts (hOBs). Exposure of hOBs to E2 (10(-8) M) 60 min before GH (5 ng/ml) significantly increased phosphorylated STAT5 (P-STAT5) levels compared with GH alone. E2 per se had no effect on P-STAT5. E2-enhanced GH signaling was effective in increasing osteopontin, bone-sialoprotein, and IGF II mRNA expression to a greater extent than GH alone. We then studied the effect of E2 on the protein levels of the negative regulator of GH signaling, suppressor of cytokine signaling-2 (SOCS2). E2 (10(-11) M-10(-7) M) reduced dose-dependently SOCS2 protein levels without modifying its mRNA expression. The silencing of SOCS2 gene prevented E2 positive effect on GH induced P-STAT5 and on GH induced bone-sialoprotein and osteopontin mRNA expression. Treatment with the inhibitor of DNA-dependent RNA synthesis, actinomycin-D, did not prevent E2 induced decrease of SOCS2, thus suggesting a non-genomic effect. E2 promoted an increase in SOCS2 ubiquitination. To determine if increased ubiquitination of SOCS2 by E2 led to degradation by proteasome, hOBs were pretreated with the proteasome inhibitor MG132 (5 μM) which blocked E2 reduction of SOCS2. These findings demonstrate for the first time that E2 can amplify GH intracellular signaling in hOBs with an essential role played by the reduction of the SOCS2 mediated feedback loop. Copyright © 2013 Elsevier Inc. All rights reserved.

A specific cementum attachment protein (CAP) was identified in human cementum and found to bind with high affinity to non-demineralized root surfaces, hydroxyapatite and fibronectin. Attempting to elucidate the biological function of this protein and its possible role in cementogenesis the capacity of CAP to promote selective cell migration towards and attachment of various periodontal derived cell populations to root surfaces in vitro was assessed. Human gingival fibroblasts (HGF), periodontal ligament cells (HPC), and alveolar bone cells (HABC) were labeled with [3H]Thymidine during their exponential growth phase. Root slices, 300 microns thick, were incubated with increasing concentrations of CAP. Untreated and fibronectin (FN) treated root slices served as negative and positive controls, respectively. Migration was assessed by placing root slices on confluent layers of labeled cells maintained in serum free medium and determining the number of cells migrated onto the root surface 3 days thereafter. Attachment was assessed by incubating root slices with labeled cell suspensions for 2 h and determining the number of attached cells. CAP promoted both cell migration and attachment dose dependently. HABC responded better than HPC and HGF to CAP treated root slices, and HPC response was higher than that of HGF. Cell attachment was dose dependently inhibited by synthetic RGD peptides. FN did not affect the migration of HGF, barely enhanced that of HABC, and was less potent than CAP at enhancing the migration of HPC. FN was more effective than CAP in promoting the attachment of HGF to root slices, but it was as potent as CAP in supporting the attachment of HPC and HABC.(ABSTRACT TRUNCATED AT 250 WORDS)