Bisphosphonates (BPs) are widely used to treat several metabolic and oncological diseases affecting the skeletal system. Despite BPs\' well-known therapeutic potential, they also displayed important side effects, among which is BPs-related osteonecrosis of the jaw, by targeting osteoclast activities, osteoblast, and osteocyte behavior. The aim of this study is to evaluate the biological effects of zoledronic acid (ZOL) in an in vitro model of periodontal ligament stem cells (PDLSCs) by using an experimental setting that resembles the in vivo conditions. PDLSCs were treated with different concentrations of ZOL ranging from 0.1 to 5 μM. The effects of ZOL exposure were evaluated on cell viability via 3-[4,5-Dimethylthiaoly]-2,5-diphenyltetrazolium bromide (MTT), cell cycle analysis, apoptosis detection, and immunofluorescence. Quantitative real-time polymerase chain reaction (PCR), colorimetric detection of alkaline phosphatase activity, and Alizarin Red S staining were performed to investigate the osteogenic potential of PDLSCs exposed to ZOL. MTT analysis showed that the viability of PDLSCs exposed to ZOL concentration ≥1.5 μM for 3 and 6 days was significantly lower (P < 0.001) than that of untreated cells. The percentage of apoptotic cells was significantly higher in PDLSCs exposed for 4 days to ZOL at 2 μM (P < 0.01) and 5 μM (P < 0.001) when compared to the control. Moreover, ZOL treatment (3 days) accounted for alterations in cell cycle distribution, with an increase in the proportion of cells in G0/G1 phase and a reduction in the proportion of cells in S phase. Chronic exposure (longer than 7 days) of PDLSCs to ZOL accounted for the downregulation of ALP, RUNX2, and COL1 genes at all tested concentrations, which fit well with the reduced alkaline phosphatase activity reported after 7 and 14 days of treatment. Reduced Col1 deposition in the extracellular matrix was reported after 14 days of treatment. Increased calcium deposits were observed in treated cells when compared to the control cultures. In conclusion, chronic exposure to 1 μM ZOL induced significant reduction of osteogenic differentiation, while ZOL concentrations ≥1.5 μM are required to impair PDLSCs viability and induce apoptosis.

OBJECTIVE: This study aims to compare the osteogenic differentiation capability of stem cells derived from human inflammatory periodontal ligament tissues (iPDLSCs) with those of stem cells derived from healthy periodontal ligament tissues (hPDLSCs). Both types of tissues were induced by stromal cell derived factor (SDF-1) in vitro.
METHODS: iPDLSCs and hPDLSCs were primarily cultured by tissue digestion method and purified by limited dilution cloning. The cells were passaged and identified by stem cell surface marker expression through flow cytometry. Then, we used thiazolyl blue tetrazolium bromide to detect and compare the proliferation capabilities of the iPDLSCs and hPDLSCs. Express of bone volumes were detected by alizarin red staining after SDF-1 was added to the cells. Using alkaline phosphatase, we evaluated the osteogenic differentiation capability of the cells induced by SDF-1. The expression levels of the osteogenesis-related genes of the cells induced by SDF-1 were determined by reverse transcription-polymerase chain reaction.
RESULTS: After purification, both iPDLSCs and hPDLSCs expressed stem cell markers. hPDLCSs had a higher proliferation capability than iPDLSCs. Osteogenesis-related genes had higher expression levels in the cells induced by SDF-1 than in those without induction (P<0.05). SDF-1 at 50 and 200 ng·mL⁻¹ concentration greatly affected the differen-tiation capabilities of iPDLSCs and hPDLSCs respectively.
CONCLUSIONS: iPDLSCs and hPDLSCs had osteogenic differentia-tion capability. The level of osteogenic differentiation in normal and inflamed periodontal ligament stem cells increases after SDF-1 induction.
目的 通过体外培养炎症来源的人牙周膜干细胞（iPDLSCs）和正常来源的人牙周膜干细胞（hPDLSCs），比较基质细胞衍生因子-1（SDF-1）对于两种来源细胞的成骨分化作用。方法 采用组织块酶消化法原代培养iPDLSCs 和hPDLSCs，经有限稀释法纯化，通过流式细胞仪对干细胞表面标记物检测鉴定后，对其进行成骨诱导；MTT法检测并比较SDF-1对两种来源的细胞增殖能力的影响；茜素红染色检测SDF-1作用于两种来源的细胞后钙化骨量的表达；碱性磷酸酶法比较SDF-1作用于两者的成骨分化能力；逆转录聚合酶链反应（RT-PCR）法检测SDF-1作用于两种牙周膜干细胞前后成骨相关基因表达水平的变化。结果 两种来源的牙周膜细胞经纯化后均阳性表达干细胞标记物。hPDLSCs较iPDLSCs增殖能力高；两种细胞经SDF-1成骨诱导培养后，成骨相关基因的表达水平均较诱导前明显上调（P<0.05），SDF-1在50、200 ng·mL⁻¹时分别对iPDLSCs和hPDLSCs细胞成骨分化作用最明显（P<0.05）。结论 正常来源和炎症来源的人牙周膜干细胞均具有成骨分化能力，SDF-1可增强两种来源的牙周膜干细胞的成骨分化能力。.

OBJECTIVE: In the present study, the early stages of in vitro bone formation in collagenated porcine scaffolds cultured with human periodontal ligament cells were investigated. The comparison between the osteogenic potential of this structure in basal and differentiating culture media was explored to predict the mechanism of its biological behavior as graft in human defect. Results were validated by synchrotron radiation X-Ray phase contrast computed microtomography (micro-CT). As the periodontal disease plays a key role in systemic and oral diseases, it is crucial to find advanced therapeutic clinical interventions to repair periodontal defects. This has been recently explored using cells and tissues developed in vitro that should ideally be immunologically, functionally, structurally and mechanically identical to the native tissue.
METHODS: In vitro cultures of human periodontal ligament cells, easily obtained by scraping of alveolar crestal and horizontal fibers of the periodontal ligament, were seeded on to collagenated porcine blocks constituted by natural cancellous and cortical bone. 3D images were obtained by synchrotron radiation micro-CT and processed with a phase-retrieval algorithm based on the transport of intensity equation.
RESULTS: Starting from the second week of culture, newly formed mineralized bone was detected in all the scaffolds, both in basal and differentiating media. Bone mineralization was proved to occur preferentially in the trabecular portion and in differentiating media.
CONCLUSIONS: The chosen method, supported by phase contrast micro-CT analysis, successfully and quantitatively monitored the early stages of bone formation and the rate of the bioscaffold resorption in basal and differentiating culture media.