Basic Research—Biology Fluocinolone Acetonide Promotes the Proliferation and Mineralization of Dental Pulp Cells Zhongning Liu, DDS, MS,* Ting Jiang, DDS, PhD,* Yixiang Wang, DDS, MS,† and Xinzhi Wang, DDS, PhD* Abstract Introduction: The aim of this study was to investigate Key Words the role of the steroid fluocinolone acetonide on the Dental pulp cells, fluocinolone acetonide, mineralization, proliferation proliferation and mineralization of human dental pulp cells (DPCs). The potential effect of fluocinolone aceto- he recovery of injured dental pulp is still one of the unresolved clinical problems. nide on reparative dentin formation and the recovery TWhen unexpected pulp exploration occurs, the survival of vital pulp will be more of injured dental pulp were evaluated. Methods: The difficult. Because healthy and vital pulp can promise a better prognosis of the injured proliferative effect of fluocinolone acetonide on DPCs tooth, preserving the vitality of pulp tissue is of key importance for long-time tooth pres- was analyzed by cholecystokinin octapeptide assay ervation (1). The ideal dental pulp capping agent should not only induce the formation and flow cytometry. The mineralized effect of fluocino- of reparative dentin but also inhibit inflammatory processes. The widely used capping lone acetonide was investigated by the detection of agents (ie, calcium hydroxide and mineral trioxide aggregate) are mainly focused on mineralization-related biomarkers including alkaline the closure of the exposed pulp; however, they are short of the anti-inflammation effect. phosphatase (ALP), bone sialoprotein, and osteocalcin So far, there is no ideal dental pulp capping material for the repair of slightly inflam- by using ALP histochemical staining, ALP activity, immu- matory pulp cases. Using anti-inflammatory medicine may be an alternative approach nostaining, alizarin red staining, and reverse- for the contaminated or mild infected pulp tissues to recover. transcriptase polymerase chain reaction. The molecules, The commonly used anti-inflammatory agents include steroidal and nonsteroidal including dentin sialophosphoprotein and Wnt4, medicines. One representative nonsteroid medicine is aspirin. Recently, it has been involved in the process of mineralization were detected shown that aspirin can improve the bone marrow mesenchymal stem cells–based cal- by real-time polymerase chain reaction and Western blot varial repair by reducing the release of interferon g or tumor necrosis factor a and analysis. Results: Low concentrations of fluocinolone therefore increases the survival of bone marrow mesenchymal stem cells (2). As the acetonide (0.1–40 mmol/L) promoted the proliferation of representation of steroid medicines, fluocinolone acetonide was primarily used in DPCs. The flow cytometry results showed that the dermal and mucosal disorders and has the potential to inhibit the release of interleukin CD146-positive subpopulation of DPCs was significantly 1a, tumor necrosis factor a, and interferon g (3–6). The first reported case of using increased after treatment with fluocinolone acetonide at steroidal medicines in dental pulp capping was performed in 1958 (7), whereas there 1and10mmol/L for 48 hours, respectively. The messenger were only a few reports relating to steroidal medicines used in dental pulp treatment. In RNA expression and activity of the early-stage mineraliza- recent years, steroids were systemically applied to endodontic treatment (8–10). The tion marker ALP were evidently increased in fluocinolone literature showed that fluocinolone acetonide can promote the proliferation and acetonide–treated DPCs compared with the untreated extracellular matrix formation in human dental pulp cells (DPCs) (11). However, control group, so did the middle-stage mineralization the role of fluocinolone acetonide in regulating pluripotent stem cell–mediated miner- marker bone sialoprotein and the late-stage mineraliza- alization, especially for dental pulp stem cells, remains unknown. In this study, we tion marker osteocalcin. Meanwhile, Wnt4 and the investigated the effect of fluocinolone acetonide on the mineralization of DPCs and dentin-specific marker dentin sialophosphoprotein were the underlying mechanism involved. obviously up-regulated by fluocinolone acetonide compared with the untreated controls. Conclusions: Materials and Methods Fluocinolone acetonide can promote the proliferation of Isolation and Culture of Human DPCs DPCs, especially for the CD146+ subpopulation. Fluocino- The study protocol was approved by the Ethics Committee of the Peking University lone acetonide can initiate the mineralization of DPCs and Health Science Center, Beijing, China. DPCs were isolated from healthy permanent has the potential role in repairing injured pulp tissues. (J premolars extracted for orthodontic reasons at the Department of Oral and Maxillo- Endod 2013;39:217–222) facial Surgery, Peking University School and Hospital of Stomatology (18- to 28-year-old donors). Briefly, pulp tissues were digested in a mixture of 3 mg/mL From the *Department of Prosthodontics and †Central Laboratory, Peking University School and Hospital of Stomatology, Beijing, China. Supported by the National Natural Science Foundation of China (30973353). Address requests for reprints to Dr Ting Jiang, Department of Prosthodontics, Peking University School and Hospital of Stomatology, 22 Zhongguancun Avenue South, Haidian District, Beijing 100081, PR China. E-mail address: [email protected]; or Dr. Yixiang Wang, Central Laboratory, Peking University School and Hospital of Stomatology, 22 Zhongguancun Avenue South, Haidian District, Beijing 100081, PR China. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2013 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2012.09.012 JOE — Volume 39, Number 2, February 2013 Fluocinolone Acetonide Promotes Proliferation, Mineralization of DPCs 217 Basic Research—Biology collagenase type I (Sigma-Aldrich, St Louis, MO) and 4 mg/mL dispase (Jiancheng, Nanjing, China), respectively, according to the manufac- (Roche, Indianapolis, IN) for 1 hour at 37C. Then, the DPCs’ suspen- turers’ protocols. sions were passed through a 70-mm strainer to obtain separated dental pulp cells (12). The single-cell suspensions were inoculated in Dul- Immunostaining of Osteogenesis-related Biomarkers becco modified Eagle medium (Gibco, Gaithersburg, MD) containing The DPCs were seeded into 12-well plates (6 Â 104 cells/well). On 10% fetal bovine serum (Hyclone, Logan, UT) (growth medium) and the following day, the cells were treated with fluocinolone acetonide at incubated at 37 C under 5% CO2. The fourth to sixth passage of DPCs the final concentration of 1 and 10 mmol/L and continued to culture for was used for further experiments. an additional 7 days for immunostaining. DPCs were treated with oste- ogenic medium as the positive control. The DPCs were washed 3 times Cell Proliferation Assay with PBS, fixed in 4% paraformaldehyde (Sigma-Aldrich) for 20 Cell proliferation was measured by cholecystokinin octapeptide minutes, and rinsed 3 times with PBS. Then, the procedure was per- (CCK-8). This method was applied according to the manufacturer’s formed according to the protocol for the SP immunohistochemical instructions. Briefly, 5 Â 103 DPCs were plated in 100 mL growth kit and the 3, 3’-diaminobenzidine (DAB) coloration kit (Zhongshan medium in a 96-well plate. After an overnight incubation, cells were Bioengineering Co Ltd, Beijing, China). The primary antibodies were treated with fluocinolone acetonide (Sigma-Aldrich) at series of antihuman bone sialoprotein (BSP, Santa Cruz, CA) and antihuman os- concentrations (0, 0.1, 1, 10, 20, 40, 60, and 100 mmol/L) for an addi- teocalcin (OCN), which was kindly provided by Dr Larry Fisher tional 24 hours. Then, 10 mL CCK-8 solution was added to each well, and (National Institute of Dental and Craniofacial Research, National Insti- the cultures were incubated for 4 hours at 37C. Cell viability was tutes of Health, Bethesda, MD), respectively. The immunostaining measured by conversion of the Dojindo highly water-soluble tetrazolium results were observed with a light microscope equipped with a camera salt WST-8 to a yellow-colored water-soluble formazan. The amount of (BX51 microscope and DP72 camera; Olympus, Tokyo, Japan) after the formazan dye generated by the activity of mitochondrial dehydroge- samples dealt with DAB color development substrate and hematoxylin nases in cells was directly proportional to the number of living cells. counterstain. Color development was quantified photometrically at 450 nm using an ELx808 Absorbance Microplate Reader (BioTek Instruments, Wi- Alizarin Red Staining nooski, VT). Viability is given in percent of the control value. The DPCs were seeded into 12-well plates (6 Â 104 cells/well). On the following day, the cells were treated with fluocinolone acetonide at Flow Cytometry Assay the final concentration of 1 and 10 mmol/L and continued to culture for The DPCs were seeded into 6-well plates (2 Â 105 cells/well). On additional 21 days for alizarin red staining. The cells were fixed in 4% the following day, the cells were treated with fluocinolone acetonide at paraformaldehyde (Sigma-Aldrich) for 20 minutes and then stained the final concentration of 1 and 10 mmol/L and continued to culture using alizarin red (Sigma-Aldrich). for additional 7 days. Then, the cells were trypsinized, rinsed with phosphate-buffered saline (PBS), and labeled with fluorescein