Jong-Hyung Lim
| Jong-Hyung Lim |
Jong-Hyung Lim, Tetsuhiro Kajikawa, Hui Wang, Xiaofei Li, George Hajishengallis
Department of Basic and Translational Sciences, University of Pennsylvania, School of Dental Medicine
Introduction
Periodontal ligament mesenchymal stem cells (referred to as PDL-MSC) have the ability to proliferate and undergo osteogenic differentiation. Aging-associated pathologies, including periodontitis involve dysfunction of PDL-MSCs. The homeostatic protein DEL-1 is highly expressed by MSCs, and Del-1 expression is downregulated in aging tissues in both humans and mice. Here, we investigated, whether Del-1 can promote proliferation and osteogenic differentiation of PDL-MSCs, thereby leading to increased bone regeneration during resolution of experimental periodontitis in aging.
Methods
To study bone tissue repair in the resolving phase of periodontitis, 12-month-old of WT C57BL/6 mice were subjected to LIP with or without ligature removal. Aged mice were euthanized after 10 days of continuous presence of ligatures (10d L), and the ligatures were removed at day 10 to allow 5 days of resolution (10d L+5d R). Mice were daily microinjected (at days 10 to 14) with DEL-1-Fc (1ug) and an equal molar amount of Fc control (0.33 µg). Periodontal bone loss was assessed morphometrically in defleshed maxillae using a dissecting microscope. mRNA expression for IL-6, IL-17a, IL-1b, TNFa, TGFb1, RANKL, and OPG in gingival tissues was analyzed by quantitative PCR. Leukocytes migrated into gingival tissue were assessed by flow cytometric analysis. For IL-17 producing cells analysis, leukocytes isolated from gingival tissues of LIP mice were reactivated with PMA and ionomycin and assessed IL-17 producing CD4+ T cells or gdT cells by flow cytometric analysis. MSCs were isolated from bone marrow and used for osteogenic differentiation and proliferation assay. For osteogenic differentiation, BMSCs were cultured in osteogenic medium. Alizarin Red staining was performed to visualize calcium nodule accumulation in MSCs, and the quantification of the released Alizarin Red was performed using a solution of 10% acetic acid. For proliferation, Ki67+ MSCs was determined as proliferating cells by flow cytometric analysis. Primary bone marrow MSCs were determined as CD29+ScaI+CD45-CD31-Ter119- cells as assessed by flow cytometric analysis.
Results
We found that local administration of exogenous DEL-1-Fc to 12-mo-old mice during the resolution of ligatured-induced periodontitis promotes bone gain compared to Fc ctrl group. Administration of DEL-1 significantly decreased IL-17 mRNA expression in gingival tissues, whereas IL-6, IL-17, IL-1b, TNFa, TGFb1, RANKL, or OPG expression was not changed. Interestingly, immunohitochemical staining detected Del-1 expression in periodontal ligament where osteogenic progenitor cells such as MSCs are localized. In in vitro experiments, we found that treatment of DEL-1-Fc promotes osteogenic differentiation of MSCs compared to Fc ctrl; consistently, Del-1-deficient MSCs were associated with defective osteogenesis compared to Del-1-sufficient MSCs. In addition, we found higher Ki67+ MSCs in DEL-1-Fc-treated group compared to Fc treated group, indicating that DEL-1 promotes MSC proliferation in vitro.
Conclusion
DEL-1 appears to promote proliferation and osteogenic differentiation of PDL-MSCs and thereby leading to alveolar bone regeneration in aging-associated periodontitis.