Effects of small molecule biochanin A on osteogenic differentiation of human dental pulp stem cells

doi:10.19438/j.cjoms.2021.03.004

Abstract

Abstract: PURPOSE: To study the effects of different concentrations of biochanin A (BCA) on osteogenic differentiation of human dental pulp stem cells (hDPSCs) and its related molecular mechanisms. METHODS: Primary human dental pulp stem cells were isolated and cultured by tissue explant method, cell phenotypes were identified by flow cytometry. Effects of different concentrations of BCA on proliferation of hDPSCs were detected by CCK-8 method, and effects of different concentrations of BCA on osteogenic differentiation of hDPSCs were analyzed by q-PCR, alkaline phosphatase (ALP) staining, and alizarin red staining. Finally, the influence of BCA on MAPK signaling pathway during osteogenic differentiation of hDPSCs was investigated through Western blot. SPSS 16.0 software package was used for statistical analysis of the data. RESULTS: hDPSCs obtained in the experiment were conformed to have the characteristics of mesenchymal stem cells by flow cytometry. CCK-8 results showed that BCA below 25 μmol/L had no effect on cell activity of hDPSCs, while over 30 μmol/L BCA inhibited the proliferation of hDPSCs. 10 μmol/L BCA significantly increased the expression of osteogenic-related genes ALP, COL-1, OPN and OCN. The staining of ALP and alizarin red was also more obvious than that of the control group and the other concentration groups. Western blot displayed that BCA activated P38/MAPK signaling pathway, but had no obvious effect on ERK and JNK pathway. CONCLUSIONS: Tissue explant method can obtain hDPSCs with higher purity. BCA may promote osteogenic differentiation of hDPSCs by activating P38/MAPK signaling pathway.

Key words: Biochanin A, Human dental pulp stem cells, Osteogenic differentiation, MAPK signaling pathway

CLC Number:

Q254

XIE Xin-ru, RAN Xue-hui, XU Wei-feng, LIU Xiu-ming, ZHANG Xiang-kai, CAO Ling-yan, ZHANG Shan-yong. Effects of small molecule biochanin A on osteogenic differentiation of human dental pulp stem cells[J]. China Journal of Oral and Maxillofacial Surgery, 2021, 19(3): 205-212.

References

[1] Zakrzewski W, Dobrzynski M, Rybak Z, et al.Selected nanomaterials' application enhanced with the use of stem cells in acceleration of alveolar bone regeneration during augmentation process[J]. Nanomaterials (Basel), 2020, 10(6): 1216-1244.
[2] Gronthos S, Mankani M, Brahim J, et al.Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo[J].Proc Natl Acad Sci USA, 2000, 97(25): 13625-13630.
[3] Tsutsui TW.Dental pulp stem cells: advances to applications[J].Stem Cells Cloning, 2020, 13: 33-42.
[4] Rapino M, Di Valerio V, Zara S, et al.Chitlac-coated thermosets enhance osteogenesis and angiogenesis in a co-culture of dental pulp stem cells and endothelial cells[J]. Nanomaterials (Basel), 2019, 9(7): 928-940.
[5] Lieberman JR, Daluiski A, Einhorn TA.The role of growth factors in the repair of bone. Biology and clinical applications[J].J Bone Joint Surg Am, 2002, 84(6): 1032-1044.
[6] 沈紫芸, 范震. 小分子化合物在骨缺损修复中对成骨分化的调控[J].口腔医学, 2019, 39(8): 739-743.
[7] 程海林, 王雷, 罗晴, 等. 鹰嘴豆芽素A基础药理作用研究进展[J].辽宁中医药大学学报, 2014, 16(12): 92-95.
[8] 周延萌, 宁慧娴, 张小敏, 等. 鹰嘴豆芽素A对雌激素缺乏诱发大鼠骨质疏松症的作用[J]. 中国药理学通报, 2014, 30(12): 1775-1776.
[9] Liao S, Feng W, Liu Y, et al.Inhibitory effects of biochanin A on titanium particle-induced osteoclast activation and inflammatory bone resorption via NF-κB and MAPK pathways[J]. J Cell Physiol, 2021, 236(2): 1432-1444.
[10] 余倩, 宋双红, 李翠芹. 鹰嘴豆芽素A和大豆苷元对MC3T3-E1细胞增殖、分化和矿化的影响[J].中药材, 2015, 38(10): 2156-2159.
[11] Liao HT, Chen CT.Osteogenic potential: comparison between bone marrow and adipose-derived mesenchymal stem cells[J].World J Stem Cells, 2014, 6(3): 288-295.
[12] Larijani B, Esfahani EN, Amini P, et al.Stem cell therapy in treatment of different diseases[J].Acta Med Iran, 2012, 50(2):79-96.
[13] Estrela C, de Alencar AH, Kitten GT, et al. Mesenchymal stem cells in the dental tissues: perspectives for tissue regeneration[J].Braz Dent J, 2011, 22(2): 91-98.
[14] Tatullo M, Marrelli M, Paduano F.The regenerative medicine in oral and maxillofacial surgery: the most important innovations in the clinical application of mesenchymal stem cells[J].Int J Med Sci, 2015, 12(1): 72-77.
[15] Dominici M, Le Blanc K, Mueller I, et al.Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement[J].Cytotherapy, 2006, 8(4): 315-317.
[16] Calloni R, Cordero EA, Henriques JA, et al.Reviewing and updating the major molecular markers for stem cells[J].Stem Cells Dev, 2013, 22(9): 1455-1476.
[17] Hankenson KD, Gagne K, Shaughnessy M.Extracellular signaling molecules to promote fracture healing and bone regeneration[J].Adv Drug Deliv Rev, 2015, 94: 3-12.
[18] Awale G, Wong E, Rajpura K, et al.Engineered bone tissue with naturally-derived small molecules[J]. Curr Pharm Des, 2017, 23(24):3585-3594.
[19] Vimalraj S, Rajalakshmi S, Raj Preeth D, et al.Mixed-ligand copper(Ⅱ) complex of quercetin regulate osteogenesis and angiogenesis[J].Mater Sci Eng C Mater Biol Appl, 2018, 83(2): 187-194.
[20] Wang Z, Wang D, Yang D, et al.The effect of icariin on bone metabolism and its potential clinical application[J]. Osteoporos Int, 2018, 29(3): 535-544.
[21] Sehm T, Fan Z, Weiss R, et al.The impact of dietary isoflavonoids on malignant brain tumors[J]. Cancer Med, 2014, 3(4): 865-877.
[22] Zakłos-Szyda M, Budryn G, Grzelczyk J, et al.Evaluation of isoflavones as bone resorption inhibitors upon interactions with receptor activator of nuclear factor-κB ligand (RANKL)[J]. Molecules, 2020, 25(1): 206-225.
[23] Su SJ, Yeh YT, Su SH, et al.Biochanin a promotes osteogenic but inhibits adipogenic differentiation: evidence with primary adipose-derived stem cells[J]. Evid Based Complement Alternat Med, 2013: 846039.
[24] Wu DQ, Zhong HM, Ding QH, et al.Protective effects of biochanin A on articular cartilage: in vitro and in vivo studies[J]. BMC Complement Altern Med, 2014,14: 444-453.
[25] Mizuno M, Fujisawa R, Kuboki Y.Type I collagen-induced osteoblastic differentiation of bone-marrow cells mediated by collagen-alpha 2 beta1 integrin interaction[J]. J Cell Physiol, 2000, 184(2): 207-213.
[26] Giachelli CM, Steitz S.Osteopontin: a versatile regulator of inflammation and biomineralization[J]. Matrix Biol, 2000, 19(7): 615-622.
[27] Roach HI.Why does bone matrix contain non-collagenous proteins? The possible roles of osteocalcin, osteonectin, osteopontin and bone sialoprotein in bone mineralisation and resorption[J]. Cell Biol Int, 1994, 18(6): 617-628.
[28] Tsao YT, Huang YJ, Wu HH, et al.Osteocalcin mediates biomineralization during osteogenic maturation in human mesenchymal stromal cells[J]. Int J Mol Sci, 2017, 18(1): 159-172.
[29] Johnson GL, Lapadat R.Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases[J]. Science, 2002, 298(5600): 1911-1912.
[30] Chen Q, Shou P, Zhang L, et al.An osteopontin-integrin interaction plays a critical role in directing adipogenesis and osteogenesis by mesenchymal stem cells[J]. Stem Cells, 2014, 32(2): 327-337.
[31] Tsai KS, Kao SY, Wang CY, et al.Type I collagen promotes proliferation and osteogenesis of human mesenchymal stem cells via activation of ERK and Akt pathways[J]. J Biomed Mater Res A, 2010, 94(3): 673-682.
[32] Kim JM, Yang YS, Park KH, et al.The ERK MAPK pathway is essential for skeletal development and homeostasis[J]. Int J Mol Sci, 2019, 20(8):1803-1818.
[33] Thouverey C, Caverzasio J. Focus on the p38 MAPK signaling pathway in bone development and maintenance [J]. Bonekey Rep, 2015,4: 711.e1-711.e8.
[34] Yamamoto N, Otsuka T, Kuroyanagi G, et al. Resveratrol reduces prostaglandin E1-stimulated osteoprotegerin synthesis in osteoblasts: suppression of stress-activated protein kinase/c-Jun N-terminal kinase [J]. Prostaglandins Other Lipid Mediat, 2015, 116-117: 57-63.
[35] Cuadrado A, Nebreda AR.Mechanisms and functions of p38 MAPK signalling[J]. Biochem J, 2010, 429(3): 403-417.