Effects of iRoot BP Plus combined with Yunnan Baiyao on mineralization of dental pulp stem cells under inflammatory conditions

doi:10.12307/2023.606

Abstract

Abstract: BACKGROUND: As a permanent preservation of living pulp, pulpotomy has been gradually applied in clinical treatment. The selection and application of pulpotomy materials can greatly enhance its clinical efficacy and expand its application range.
OBJECTIVE: To study the effects of Yunnan Baiyao, iRoot BP Plus and their combination on the mineralization of pulp stem cells under the condition of inflammation.
METHODS: Human primary dental pulp stem cells were cultured by tissue block method, and the effects of Yunnan Baiyao and iRoot BP Plus on the proliferation of dental pulp stem cells were determined by MTT assay to determine the optimal concentration of intervention quality. Passages 3-6 dental pulp stem cells were divided into five groups: normal control group, inflammatory control group, Yunnan Baiyao group, iRoot BP Plus group, and Yunnan Baiyao + iRoot BP Plus group. Cells in the inflammatory control group, Yunnan Baiyao group, iRoot BP Plus group, and Yunnan Baiyao + iRoot BP Plus group were treated with 1 μg/mL lipopolysaccharide for 2 hours to establish models and then stimulated with 75 μg/mL Yunnan Baiyao, 100 μg/mL iRoot BP Plus alone and their combination. Effects of drugs on cellular mineralization were determined by alkaline phosphatase assay, alizarin red S staining, real-time-PCR and western blot assay.
RESULTS AND CONCLUSION: (1) Compared with the inflammatory control group, Yunnan Baiyao, iRoot BP Plus and their combination inhibited the mRNA expression of tumor necrosis factor-α (P < 0.001). The mRNA expression of tumor necrosis factor-α in Yunnan Baiyao + iRoot BP Plus group was significantly lower than that in Yunnan Baiyao group and iRoot BP Plus group (P < 0.001). (2) Compared with the inflammatory control group, the number of mineralized nodules and alkaline phosphatase activity were significantly increased after Yunnan Baiyao, iRoot BP Plus, and their combined application. The number of mineralized nodules and alkaline phosphatase activity in Yunnan Baiyao + iRoot BP Plus group were higher than those in Yunnan Baiyao and iRoot BP Plus groups (P < 0.001). (3) Compared with the inflammatory control group, the mRNA expression levels of DSPP, IBSP and MEPE increased after Yunnan Baiyao, iRoot BP Plus, and their combined application (P < 0.001). The mRNA expression levels of DSPP, IBSP, and MEPE in Yunnan Baiyao + iRoot BP Plus group were higher than those in Yunnan Baiyao group and iRoot BP Plus group (P < 0.001). (4) Compared with the inflammatory control group, phosphorylation of Akt and mTOR was increased in Yunnan Baiyao group and Yunnan Baiyao + iRoot BP Plus group (P < 0.001). (5) The results show that both Yunnan Baiyao and iRoot BP Plus can promote the mineralization of dental pulp stem cells, and the combined application of Yunnan Baiyao and iRoot BP Plus has a more significant mineralization effect. The effect of Yunnan Baiyao on promoting the mineralization of dental pulp stem cells is associated with the Akt/mTOR pathway.

Key words: pulpitis, Yunnan Baiyao, iRoot BP Plus, pulpotomy, inflammation, mineralization

CLC Number:

Zhao Ying, Liang Guangzhi, Zhou Yuqi, Wang Tianqi, Liu Yunxia, Liu Xiaoying, Zhang Juanjuan, Sun Yan. Effects of iRoot BP Plus combined with Yunnan Baiyao on mineralization of dental pulp stem cells under inflammatory conditions[J]. Chinese Journal of Tissue Engineering Research, 2023, 27(19): 2960-2967.

Figures/Tables 8

随着时间的增加，不同质量浓度云南白药组的牙髓干细胞相对数量均增加。第1天，当云南白药质量浓度≤75 μg/mL时，随着云南白药质量浓度的升高细胞数量无明显变化，当云南白药质量浓度为100 μg/mL时，细胞数量有所升高；第3天，当云南白药质量浓度≤75 μg/mL时，随着云南白药质量浓度的升高细胞数量无明显变化，当云南白药质量浓度为100 μg/mL时，细胞数量出现下降；第5天，当云南白药质量浓度≤50 μg/mL时，细胞数量略有降低，而云南白药质量浓度为50-75 μg/mL时，细胞数量随着云南白药质量浓度的增加而升高，而在云南白药质量浓度为100 μg/mL时，细胞数量出现明显下降，即当云南白药浸提液质量浓度为75 μg/mL时，药物对牙髓干细胞增殖的影响最小，细胞毒性小，因而在后续实验中，云南白药浸提液选用75 μg/mL质量浓度。随着时间增加，不同质量浓度iRoot BP Plus浸提液组细胞数量同样整体呈增加趋势。第1天和第3天，当iRoot BP Plus浸提液质量浓度≤100 μg/mL时，细胞数量随iRoot BP Plus质量浓度升高而增加，当iRoot BP Plus质量浓度≥100 μg/mL时，细胞数量无明显变化；在第5天，在质量浓度≤100 μg/mL时细胞数量随着iRoot BP Plus质量浓度升高而增加，当质量浓度为150 μg/mL时，细胞数量未见继续增加，且当质量浓度升至200 μg/mL时，细胞数量反而较之前下降，通过上述实验结果，选择质量浓度为100 μg/mL的iRoot BP Plus浸提液应用于后续实验。 2.3 云南白药及iRoot BP Plus浸提液对脂多糖诱导下牙髓干细胞炎症的影响炎症对照组肿瘤坏死因子α mRNA相对表达量均高于正常对照组(P < 0.001)，表明经脂多糖处理后细胞进入炎症状态，细胞炎症模型建立成功。经云南白药、iRoot BP Plus及其联合应用处理1，3 d后，肿瘤坏死因子α mRNA相对表达量均低于炎症对照组(P < 0.001)，表明上述2种药物对炎症相关因子肿瘤坏死因子α mRNA均具有抑制作用，在上述2个时间点云南白药组对肿瘤坏死因子α mRNA相对表达量的抑制作用明显强于iRoot BP Plus组，云南白药+iRoot BP Plus组对肿瘤坏死因子α mRNA相对表达量的抑制作用强于2个单独处理组。初步认定云南白药对牙髓干细胞炎症状态的抑制作用较iRoot BP Plus具有明显优势，且2种药物具有协同作用，见图4。"

References

[1] LINAS N, DECERLE N, MUNOZ-SANCHEZ ML, et al. Long-term Outcomes of Full Pulpotomy in Permanent Molars for Patients Treated in a Single, Short Session under Special Conditions. J Endod. 2020;46(11):1597-1604.
[2] TAN SY, YU VSH, LIM KC, et al. Long-term Pulpal and Restorative Outcomes of Pulpotomy in Mature Permanent Teeth. J Endod. 2020;46(3):383-390.
[3] ZANINI M, HENNEQUIN M, COUSSON PY. A Review of Criteria for the Evaluation of Pulpotomy Outcomes in Mature Permanent Teeth. J Endod. 2016;42(8):1167-1174.
[4] CUSHLEY S, DUNCAN HF, LAPPIN MJ, et al. Pulpotomy for mature carious teeth with symptoms of irreversible pulpitis: A systematic review. J Dent. 2019;88:103158.
[5] LUIZ DE OLIVEIRA DA ROSA W, MACHADO DA SILVA T, FERNANDO DEMARCO F, et al. Could the application of bioactive molecules improve vital pulp therapy success? A systematic review. J Biomed Mater Res A. 2017;105(3):941-956.
[6] PEZELJ-RIBARIC S, ANIC I, BREKALO I, et al. Detection of tumor necrosis factor alpha in normal and inflamed human dental pulps. Arch Med Res. 2002;33(5):482-484.
[7] SILVA AC, FARIA MR, FONTES A, et al. Interleukin-1 beta and interleukin-8 in healthy and inflamed dental pulps. J Appl Oral Sci. 2009;17(5):527-532.
[8] RAO Q, KUANG J, MAO C, et al. Comparison of iRoot BP Plus and Calcium Hydroxide as Pulpotomy Materials in Permanent Incisors with Complicated Crown Fractures: A Retrospective Study. J Endod. 2020;46(3):352-357.
[9] LU J, LI Z, WU X, et al. iRoot BP Plus promotes osteo/odontogenic differentiation of bone marrow mesenchymal stem cells via MAPK pathways and autophagy. Stem Cell Res Ther. 2019;10(1):222.
[10] CHEN X, ZHANG H, ZHONG J, et al. Comparison of indirect pulp treatment and iRoot BP Plus pulpotomy in primary teeth with extremely deep caries: a prospective randomized trial. Clin Oral Investig. 2021;25(5):3067-3076.
[11] ABOU ELREASH A, HAMAMA H, GRAWISH M, et al. A laboratory study to test the responses of human dental pulp stem cells to extracts from three dental pulp capping biomaterials. Int Endod J. 2021;54(7):1118-1128.
[12] LI R, ALEX P, YE M, et al. An old herbal medicine with a potentially new therapeutic application in inflammatory bowel disease. Int J Clin Exp Med. 2011;4(4):309-319.
[13] TANG ZL, WANG X, YI B, et al. Effects of the preoperative administration of Yunnan Baiyao capsules on intraoperative blood loss in bimaxillary orthognathic surgery: a prospective, randomized, double-blind, placebo-controlled study. Int J Oral Maxillofac Surg. 2009;38(3):261-266.
[14] 黎慧,于浩滢,杨小佳,等.云南白药提取物对大鼠实验性牙周炎的治疗作用[J].华西药学杂志,2018,33(6):472-474.
[15] WANG S, JIAO J, DONG X, et al. Effect of total knee replacement combined with Yunnan Baiyao on the expression of serum IL-6, TNF-α, VCAM-1 and MMP-9 in patients with knee osteoarthritis. Panminerva Med. 2020 Aug 5. doi: 10.23736/S0031-0808.20.04016-1. Online ahead of print.
[16] GRONTHOS S, MANKANI M, BRAHIM J, et al. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci U S A. 2000;97(25): 13625-13630.
[17] MIURA M, GRONTHOS S, ZHAO M, et al. SHED: stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci U S A. 2003;100(10): 5807-5812.
[18] ABD-ELMEGUID A, YU DC, KLINE LW, et al. Dentin matrix protein-1 activates dental pulp fibroblasts. J Endod. 2012;38(1):75-80.
[19] SILLER AF, WHYTE MP. Alkaline Phosphatase: Discovery and Naming of Our Favorite Enzyme. J Bone Miner Res. 2018;33(2):362-364.
[20] HALLING LINDER C, EK-RYLANDER B, KRUMPEL M, et al. Bone Alkaline Phosphatase and Tartrate-Resistant Acid Phosphatase: Potential Co-regulators of Bone Mineralization. Calcif Tissue Int. 2017;101(1):92-101.
[21] CHAUDHARY SC, KUZYNSKI M, BOTTINI M, et al. Phosphate induces formation of matrix vesicles during odontoblast-initiated mineralization in vitro. Matrix Biol. 2016;52-54:284-300.
[22] SIMÃO AM, BOLEAN M, HOYLAERTS MF, et al. Effects of pH on the production of phosphate and pyrophosphate by matrix vesicles’ biomimetics. Calcif Tissue Int. 2013;93(3):222-232.
[23] MALAVAL L, AUBIN JE, VICO L. Role of the small integrin-binding ligand N-linked glycoprotein (SIBLING), bone sialoprotein (BSP) in bone development and remodeling. Osteoporos Int. 2009;20(6):1077-1080.
[24] ZHANG YL, YIN JH, DING H, et al. Protective effect of VK2 on glucocorticoid-treated MC3T3-E1 cells. Int J Mol Med. 2017;39(1):160-166.
[25] ZOCH ML, CLEMENS TL, RIDDLE RC. New insights into the biology of osteocalcin. Bone. 2016;82:42-49.
[26] AN Y, SONG Y, WANG Z, et al. Effect of low-intensity pulsed ultrasound on the biological behaviors of bone marrow mesenchymal stem cells on titanium with different surface topographies. Am J Transl Res. 2018;10(1):67-76.
[27] RAHMAN SU, OH JH, CHO YD, et al. Fibrous Topography-Potentiated Canonical Wnt Signaling Directs the Odontoblastic Differentiation of Dental Pulp-Derived Stem Cells. ACS Appl Mater Interfaces. 2018;10(21):17526-17541.
[28] BERTACCHINI J, HEIDARI N, MEDIANI L, et al. Targeting PI3K/AKT/mTOR network for treatment of leukemia. Cell Mol Life Sci. 2015;72(12):2337-2347.
[29] MANFREDI GI, DICITORE A, GAUDENZI G, et al. PI3K/Akt/mTOR signaling in medullary thyroid cancer: a promising molecular target for cancer therapy. Endocrine. 2015;48(2):363-370.
[30] FERRERO H. Commentary on regulation of leiomyoma stem cell proliferation by WNT4 via AKT-dependent β-Catenin activation. Fertil Steril. 2020;114(6): 1192-1193.
[31] JIANG Y, LIU F, ZOU F, et al. PBX homeobox 1 enhances hair follicle mesenchymal stem cell proliferation and reprogramming through activation of the AKT/glycogen synthase kinase signaling pathway and suppression of apoptosis. Stem Cell Res Ther. 2019;10(1):268.
[32] LEI X, JIAO J. UTX Affects Neural Stem Cell Proliferation and Differentiation through PTEN Signaling. Stem Cell Reports. 2018;10(4):1193-1207.
[33] RUSSELL RC, FANG C, GUAN KL. An emerging role for TOR signaling in mammalian tissue and stem cell physiology. Development. 2011;138(16): 3343-3356.
[34] ZHOU R, MA Y, QIU S, et al. Metformin promotes cell proliferation and osteogenesis under high glucose condition by regulating the ROS‑AKT‑mTOR axis. Mol Med Rep. 2020;22(4):3387-3395.
[35] ALSALLEEH F, CHUNG N, STEPHENSON L. Antifungal activity of endosequence root repair material and mineral trioxide aggregate. J Endod. 2014;40(11):1815-1819.