Sains Malaysiana 51(5)(2022): 1449-1464

http://doi.org/10.17576/jsm-2022-5105-15

Higher Concentration of Ascorbic Acid as a Sole Induction Factor for Osteogenesis on MC3T3-E1 Cell Model

(Kepekatan Tinggi Asid Askorbik sebagai Faktor Aruhan Tunggal untuk Osteogenesis pada Model Sel MC3T3-E1)

FARINAWATI YAZID¹, WAN CHING NG², NUR ATMALIYA LUCHMAN^2, SHAHRUL HISHAM ZAINAL ARIFFIN³ & ROHAYA MEGAT ABDUL WAHAB^2,*

¹Discipline of Paediatric Dentistry, Department of Family Oral Health, Universiti Kebangsaan Malaysia, Jalan Raja Muda Aziz, 50300 Kuala Lumpur, Federal Territory, Malaysia

²Discipline of Orthodontics, Department of Family Oral Health, Universiti Kebangsaan Malaysia, Jalan Raja Muda Aziz, 50300 Kuala Lumpur, Federal Territory, Malaysia

³Department of Biological Sciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan, Malaysia

Received: 31 March 2021/Accepted: 4 October 2021

Abstract

This research aimed to investigate the potential of ascorbic acid (Asc) to act independently as an osteogenic induction factor in a murine pre-osteoblast (MC3T3-E1) model. MC3T3-E1 cells were seeded in culture wells and hydroxyapatite scaffold for two-dimensional and three-dimensional analyses respectively. Cell morphology, viability, osteoblast differentiation, and mineralisation potentials of MC3T3-E1 cells were compared between induction of standard (50 µg/mL) and doubled (100 µg/mL) Asc concentrations in growth media. Cells with fibroblast-like morphology became confluent earlier on day 6 in the standard group compared to the doubled group on day 9. Cell viability and differentiation potential were significantly increased in the doubled group (p < 0.01). Mineralisation occurred in the doubled group after 15 days of seeding but no mineralisation was seen in the standard group. Findings were similar in 3D analysis whereby mineralized nodules were seen only in the doubled group. The relative expression of collagen 1(α) and osteocalcin genes were increased in the doubled group than the standard group. Doubling Asc concentration in a growth medium to 100 µg/mL can induce viability, differentiation, and mineralisation of MC3T3-E1 cells. Thus, higher concentration of ascorbic acid can potentially be used as the sole induction factor in osteogenic medium.

Keywords: Ascorbic acid; growth media; MC3T3-E1 cells; osteogenic differentiation; sole induction

Abstrak

Penyelidikan ini bertujuan untuk mengkaji potensi asid askorbik (Asc) bagi tindak balas secara bebas sebagai faktor induksi osteogenik dalam model pra-osteoblas murine (MC3T3-E1). Sel MC3T3-E1 dibenihkan dalam telaga kultur dan perancah hidroksi apatit untuk analisa dua dimensi dan tiga dimensi. Morfologi sel, kebolehidupan sel, pembezaan osteoblas dan potensi pemineralan sel MC3T3-E1 dibandingkan antara induksi kepekatan piawai (50 µg/mL) dan kumpulan dua kali ganda (100 µg/mL) kepekatan asid askorbik di dalam media pertumbuhan. Sel dengan morfologi seperti fibroblas menjadi konfluensi pada hari ke 6 dalam kumpulan piawai berbanding kumpulan dua kali ganda pada hari ke 9. Kebolehidupan sel dan potensi pembezaan meningkat dengan ketara pada kumpulan dua kali ganda (p < 0.01). Pemineralan berlaku pada kumpulan yang berlipat ganda selepas 15 hari pengkulturan tetapi tiada pemineralan dilihat pada kumpulan piawai. Penemuan yang sama dilihat dalam analisis 3D manakala nodul mineral hanya dilihat pada kumpulan dua kali ganda. Ekspresi relatif gen kolagen 1(α) dan osteokalsin meningkat pada kumpulan dua kali ganda berbanding kumpulan piawai. Menggandakan kepekatan asid askorbik dalam medium pertumbuhan hingga 100 µg/mL boleh mengaruh keviabelan, pembezaan dan pemineralan sel MC3T3-E1. Oleh itu, kepekatan asid askorbik yang lebih tinggi berpotensi digunakan sebagai faktor induksi tunggal dalam medium osteogenik.

Kata kunci: Aruhan tunggal; asid askorbik; media pertumbuhan; sel MC3T3-E1; pembezaan osteogenik

REFERENCES

Aghajanian, P., Hall, S., Wongworawat, M.D. & Mohan, S. 2015. The roles and mechanisms of actions of vitamin C in bone: New developments. J. Bone Miner. Res. 30(11): 1945-1955.

Ariffin, S.H.Z., Manogaran, T., Abidin, I., Wahab, R.M.A. & Senafi, S. 2017. A perspective on stem cells as biological systems that produce differentiated osteoblasts and odontoblasts. Curr. Stem Cell Res. Ther. 12(3): 247-259.

Chan, D., Lamande, S.R., Cole, W.G. & Bateman, J.F. 1990. Regulation of procollagen synthesis and processing during ascorbate-induced extracellular matrix accumulation in vitro. The Biochemical Journal 269(1): 175-181.

Choi, J.Y., Lee, B.H., Song, K.B., Park, R.W., Kim, I.S., Sohn, K.Y., Jo, S.J. & Ryoo, H.M. 1996. Expression patterns of bone-related proteins during osteoblastic differentiation in MC3T3-E1 cells. J. Cell Biochem. 61(4): 609-618.

Cordonnier, T., Langonné, A., Corre, P., Renaud, A., Sensebé, L., Rosset, P., Layrolle, P. & Sohier, J. 2014. Osteoblastic differentiation and potent osteogenicity of three-dimensional hBMSC-BCP particle constructs. J. Tissue Eng. Regen. Med. 8(5): 364-376.

Czekanska, E.M., Stoddart, M.J., Richards, R.G. & Hayes, J.S. 2012. In search of an osteoblast cell model for in vitro research. Eur. Cells Mater. 24: 1-17.

Farinawati, Y., Nur Atmaliya, L., Rohaya, M.A.W., Shahrul Hisham, Z.A. & Sahidan, S. 2018. Proliferation and osteoblast differentiation mice dental pulp stem cells between enzyme digestion and outgrowth method. Sains Malaysiana 47(4): 691-698.

Fernandes, G., Barone, A.W. & Dziak, R. 2017. The effect of ascorbic acid on bone cancer cells in vitro. Cogent. Biol. 3(1): 1288335.

Finck, H., Hart, A.R., Jennings, A. & Welch, A.A. 2014. Is there a role for vitamin C in preventing osteoporosis and fractures? A review of the potential underlying mechanisms and current epidemiological evidence. Nutr. Res. Rev. 27(2): 268-283.

Franceschi, R.T. & Iyer, B.S. 1992. Relationship between collagen synthesis and expression of the osteoblast phenotype in MC3T3‐E1 cells. J. Bone Miner. Res. 7(2): 235-246.

Franceschi, R.T., Iyer, B.S. & Cui, Y. 1994. Effects of ascorbic acid on collagen matrix formation and osteoblast differentiation in murine MC3T3-E1 cells. J. Bone Miner. Res. 9(6): 843-854.

Fu, C., Yang, X., Tan, S. & Song, L. 2017. Enhancing cell proliferation and osteogenic differentiation of MC3T3-E1 pre-osteoblasts by BMP-2 delivery in graphene oxide-incorporated PLGA/HA biodegradable microcarriers. Sci. Rep. 7: 1-13.

Fujisawa, K., Hara, K., Takami, T., Okada, S., Matsumoto, T., Yamamoto, N. & Sakaida, I. 2018. Evaluation of the effects of ascorbic acid on metabolism of human mesenchymal stem cells. Stem Cell Research & Therapy 9: 93.

Gerard, C. & Goldbeter, A. 2014. The balance between cell cycle arrest and cell proliferation: Control by the extracellular matrix and by contact inhibition. Interface Focus 4(3): 20130075-20130075.

Guo, J., Li, C., Zhang, Q., Wu, G., Deacon, S.A., Chen, J., Hu, H., Zou, S. & Ye, Q. 2011. Secondary bone grafting for alveolar cleft in children with cleft lip or cleft lip and palate. Cochrane Database Syst. Rev. (6): CD008050.

Hadzir, S.N., Ibrahim, S.N., Wahab, R.M.A., Abidin, I.Z.Z., Senafi, S., Ariffin, Z.Z., Razak, M.A. & Ariffin, S.H.Z. 2014. Ascorbic acid induces osteoblast differentiation of human suspension mononuclear cells. Cytotherapy 16(5): 674-682.

Hammoudeh, J.A., Fahradyan, A., Gould, D.J., Liang, F., Imahiyerobo, T. & Urbinelli. 2017. A comparative analysis of recombinant human bone morphogenetic protein-2 with a demineralized bone matrix versus iliac crest bone graft for secondary alveolar bone grafts in patients with cleft lip and palate: Review of 501 cases. Plastic Reconstructive Surgery 140(2): 318e-325e.

Harada, S.I., Matsumoto, T. & Ogata, E. 1991 Role of ascorbic acid in the regulation of proliferation in osteoblast‐like MC3T3‐El cells. J. Bone Miner. Res. 6(9): 903-908.

Hayrapetyan, A., Jansen, J.A. & Beucken, J.V.d. 2015. Signaling pathways involved in osteogenesis and their application for bone regenerative medicine. Tissue Eng. Part B Rev. 21(1): 75-87.

Hong, D., Chen, H.X., Yu, H.Q., Liang, Y., Wang, C., Lian, Q.Q., Deng, H.T. & Ge, R.S.  2010. Morphological and proteomic analysis of early stage of osteoblast differentiation in osteoblastic progenitor cells. Exp. Cell Res. 316(14): 2291-2300.

Jimi, E., Hirata, S., Osawa, K., Terashita, M., Kitamura, C. & Fukushima, H. 2012. The current and future therapies of bone regeneration to repair bone defects. Int. J. Dent. 2012: 148261.

Kodama, H., Amagai, Y., Sudo, H., Kasai, S. & Yamamoto, S. 1981. Establishment of a clonal osteogenic cell line from newborn mouse calvaria. Jap. J. Oral Biol. 23: 899-901.

Kuiper, C. & Vissers, M.C.M. 2014. Ascorbate as a co-factor for Fe- and 2-oxoglutarate dependent dioxygenases: Physiological activity in tumor growth and progression. Frontiers in Oncology 4: 359.

Kumar, P., Fathima, G. & Vinitha, B. 2013. Bone grafts in dentistry. J. Pharm. Bioallied Sci. 5(Suppl 1): S125-S127.

Langenbach, F. & Handschel, J. 2013. Effects of dexamethasone, ascorbic acid and β-glycerophosphate on the osteogenic differentiation of stem cells in vitro. Stem Cell Res. Ther. 4(5): 117.

Livak, K.J. & Schmittgen, T.D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method. Methods 25(4): 402-408.

MacIsaac, Z.M., Rottgers, S.A., Davit, A.J., Ford, M., Losee, J.E. & Kumar, A.R. 2012. Alveolar reconstruction in cleft patients: Decreased morbidity and improved outcomes with supplemental demineralized bone matrix and cancellous allograft. Plastic Reconstructive Surgery 130(3): 625-632.

Marsh, M.E., Munne, A.M., Vogel, J.J., Cui, Y. & Franceschi, R.T. 2009. Mineralization of bone-like extracellular matrix in the absence of functional osteoblasts. J. Bone Miner. Res. 10(11): 1635-1643.

Matthews, B.G., Naot, D., Callon, K.E., Musson, D.S., Locklin, R., Hulley, P.A., Grey, A. & Cornish, J. 2014. Enhanced osteoblastogenesis in three-dimensional collagen gels. Bonekey Reports 3: 560.

Motamedian, S., Tabatabaei, F., Akhlaghi, F., Torshabi, M., Gholamin, P. & Khojasteh, A. 2016. Response of dental pulp stem cells to synthetic, allograft, and xenograft bone scaffolds. Int. J. Periodont. Rest. 37(1): 47-59.

Nawi, N.S.M., Ariffin, Z., Alam, M.K., Noor, F.M. & Hassan, A. 2013. The assessment of proliferation rate of dental pulp stem cells and stem cell from human exfoliated deciduous teeth by using two different scaffolds. Int. Med. J. 20: 593-596.

Quarles, L.D., Yohay, D.A., Lever, L.W., Caton, R. & Wenstrup, R.J. 1992. Distinct proliferative and differentiated stages of murine MC3T3‐E1 cells in culture: An in vitro model of osteoblast development. J. Bone Miner. Res. 7(6): 683-692.

Roberts, T.T. & Rosenbaum, A.J. 2012. Bone grafts, bone substitutes and orthobiologics. Organogenesis 8(4): 114-124.

Ruijtenberg, S. & Heuvel, S.v.D. 2016 Coordinating cell proliferation and differentiation: Antagonism between cell cycle regulators and cell type-specific gene expression. Cell Cycle 15(2): 196-212.

Seebach, C., Schultheiss, J., Wilhelm, K., Frank, J. & Henrich, D. 2010. Comparison of six bone-graft substitutes regarding to cell seeding efficiency, metabolism, and growth behaviour of human mesenchymal stem cells (MSC) in vitro. Injury 41(7): 731-738.

Shamsuddin, S.A., Ramli, R., Razali, M., Baharin, B., Sulaiman, S., Hwei Ng, M., Low, C.K., Jabar, M.N.A., Nordin, R., Yahaya, N., Shukur, S.S.A., Siar, C.H. & Hj Idrus, R. 2017. Guided bone regeneration using autologous plasma, bone marrow cells and Β-TCP/HA granules for experimental alveolar ridge reconstruction in Macaca fascicularis. J. Bio.mater. Tissue Eng. 7(2): 111-118.

Sudo, H., Kodama, H.A., Amagai, Y., Yamamoto, S. & Kasai, S. 1983. In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria. J. Cell Biol. 96(1): 191-198.

Vater, C., Kasten, P. & Stiehler, M. 2011. Culture media for the differentiation of mesenchymal stromal cells. Acta Biomater. 7(2): 463-477.

Wahab, R.M.A., Abdullah, N., Ariffin, S.H.Z., Che Abdullah, C.A. & Yazid, F. 2020. Effects of the sintering process on nacre-derived hydroxyapatite scaffolds for bone engineering. Molecules 25(14): 3129.

Wan, H.W.N., Ghafar, N.A., Chin, K.Y. & Ima-Nirwana, S. 2018. Annatto-derived tocotrienol stimulates osteogenic activity in preosteoblastic MC3T3-E1 cells: A temporal sequential study. Drug Des. Devel. Ther. 12: 1715-1726.

Yan, X.Z., Yang, W., Yang, F., Kersten-Niessen, M., Jansen, J.A. & Both, S.K. 2014. Effects of continuous passaging on mineralization of MC3T3-E1 cells with improved osteogenic culture protocol. Tissue Eng. Part C Methods 20(3): 198-204.

Yazid, F., Kay, A., Qin, W., Luchman, N., Wahab, R.A. & Ariffin, S. 2019. Morphology and osteogenic capability of MC3T3-E1 cells on granular hydroxyapatite scaffold. J. Biol. Sci. 19: 201-209.

Yazid, M.D., Ariffin, S.H.Z., Senafi, S., Razak, M.A. & Wahab, R.M.A. 2010. Determination of the differentiation capacities of murines’ primary mononucleated cells and MC3T3-E1 cells. Cancer Cell Int. 10: 1-12.

Zakaria, Z., Seman, C.N.Z.C., Buyong, Z., Sharifudin, M.A., Zulkifly, A.H. & Khalid, K.A. 2016. Histological evaluation of hydroxyapatite granules with and without platelet-rich plasma versus an autologous bone graft: Comparative study of biomaterials used for spinal fusion in a New Zealand white rabbit model. Sultan Qaboos Univ. Med. J. 16(4): 422-429.

Zulkifly, A.H., Khalid, K.A., Azril, M.A., Shukrimi, A., Yusof, N.M. & Ahmad, A.C. 2008. Granules hydroxyapatite application in fractures. Eur. Cells Maters. 16: 56.

^*Corresponding author; email: rohaya_megat@ukm.edu.my