Sains Malaysiana 47(4)(2018): 691-698

http://dx.doi.org/10.17576/jsm-2018-4704-06

Proliferation and Osteoblast Differentiation Mice Dental Pulp Stem Cells between Enzyme Digestion and Outgrowth Method

(Proliferasi dan Pembezaan Osteoblas Sel Stem Pulpa Gigi Mencit antara Kaedah Pencernaan

Enzim dan Eksplan)

FARINAWATI YAZID^1*, NUR ATMALIYA LUCHMAN¹, ROHAYA MEGAT ABDUL WAHAB¹, SHAHRUL HISHAM ZAINAL ARIFFIN² & SAHIDAN SENAFI²

¹Faculty of Dentistry, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz,

50300 Kuala Lumpur, Federal Territory, Malaysia

²School of Bioscience and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor Darul Ehsan, Malaysia

Received: 26 July 2017/Accepted: 6 November 2017

ABSTRACT

The isolation method for dental pulp stem cells (DPSCs) is still unclear to obtain a conducive environment for DPSCs to proliferate. Enzymatic digestion and outgrowth method are two commonly used methods for DPSCs isolation but are not well characterized in mice DPSCs. This study aimed to compare these isolation methods and differentiation potential of mice DPSCs into bone cells. Dental pulp was extracted from mice’s incisors and subjected to isolation either by collagenase 1A or culture of pulp tissue in complete alpha-Modified Eagle Medium (αMEM). Both cells isolated were cultured until passage 4 and subjected to in vitro proliferation and differentiation analysis. Both cells exhibited fibroblast- liked morphology, but cells isolated by enzyme digestion proliferate faster compare to outgrowth method. After 21 days of osteoblast differentiation, DPSCs isolated from enzyme digestion method showed alkaline phosphatase (ALP) activity slightly different as compared to outgrowth method. In conclusion, there is a significant difference between the cells isolated from enzyme digestion compare to outgrowth method with regard to proliferation and osteoblast differentiation. Thus, it is preferable to isolate by enzyme digestion as it is faster and consistent compared to outgrowth method.

Keywords: Adherent cells; alkaline phosphatase; in vitro; mesenchymal stem cells

ABSTRAK

Kaedah pemencilan bagi sel stem pulpa gigi (DPSCs) masih kurang jelas terutamanya bagi mendapatkan persekitaran yang kondusif bagi DPSCs berproliferasi. Kaedah pencernaan enzim dan eksplan merupakan dua kaedah yang biasa digunakan untuk memencilkan DPSCs namun kurang dicirikan pada DPSCs mencit. Kajian ini bertujuan untuk membandingkan kaedah pemencilan dan potensi perbezaan DPSCs mencit kepada sel tulang. Pulpa gigi diekstrak daripada gigi kacip mencit dan pemencilan sel dilakukan sama ada menggunakan kolagenase 1A atau pengkulturan tisu pulpa pada medium lengkap alpha-modified eagle medium (αMEM). Kedua-dua sel yang dipencilkan dikulturkan sehingga pasaj 4 dan analisis proliferasi dan pembezaan secara in vitro dilakukan. Kedua-dua sel menunjukkan morfologi fibroblas namun sel yang diasingkan melalui pencernaan enzim berproliferasi lebih cepat berbanding dengan kaedah eksplan. Selepas 21 hari pembezaan kepada sel osteoblas, DPSCs yang dipencilkan melalui kaedah pencernaan enzim menunjukkan aktiviti alkali fosfatase (ALP) sedikit berbeza berbanding kaedah eksplan. Kesimpulannya, terdapat perbezaan yang signifikan daripada sel yang dipencilkan melalui kaedah percernaan enzim berbanding eksplan terutamanya daripada segi proliferasi dan pembezaan osteoblas. Oleh itu, adalah lebih baik untuk memencilkan sel melalui kaedah pencernaan enzim kerana ia adalah lebih cepat dan konsisten berbanding dengan kaedah eksplan.

Kata kunci: Alkali fosfatase; in vitro; sel melekat; sel stem mesenkima

REFERENCES

Akmal, M.N., Zarina, Z.I., Rohaya, M., Sahidan, S., Zaidah, Z. & Hisham, Z.S. 2014. Isolation and characterization of dental pulp stem cells from murine incisors. Journal of Biological Sciences 14(4): 327.

Bakopoulou, A., Leyhausen, G., Volk, J., Tsiftsoglou, A., Garefis, P., Koidis, P. & Geurtsen, W. 2011. Assessment of the impact of two different isolation methods on the osteo/odontogenic differentiation potential of human dental stem cells derived from deciduous teeth. Calcified Tissue International 88(2): 130-141.

Barbara, Z., Eriberto, B., Giulia, B., Letizia, F., Chiara, G., Ferrarese, N., Stefano, S. & Edoardo, S. 2011. Dental pulp stem cells and tissue engineering strategies for clinical application on odontoiatric field Ed.: INTECH Open Access Publisher.

Beck, G.R., Sullivan, E.C., Moran, E. & Zerler, B. 1998. Relationship between alkaline phosphatase levels, osteopontin expression, and mineralization in differentiating MC3T3-E1 osteoblasts. Journal of Cellular Biochemistry 68(2): 269-280.

Birmingham, E., Niebur, G., McHugh, P., Shaw, G., Barry, F.P. & McNamara, L.M. 2012. Osteogenic differentiation of mesenchymal stem cells is regulated by osteocyte and osteoblast cells in a simplified bone niche. Eur. Cell Mater. 23: 13-27.

de Souza, L.M., Bittar, J.D., da Silva, I.C.R., de Toledo, O.A., de Macedo Brígido, M. & Fonseca, M.J.P. 2015. Comparative isolation protocols and characterization of stem cells from human primary and permanent teeth pulp. Brazilian Journal of Oral Sciences 9(4): 427-433.

Djouad, F., Jackson, W.M., Bobick, B.E., Janjanin, S., Song, Y., Huang, G. & Tuan, R.S. 2010. Activin A expression regulates multipotency of mesenchymal progenitor cells. Stem Cell Res. Ther. 1(2): 11.

Dominici, M., Le Blanc, K., Mueller, I., Slaper-Cortenbach, I., Marini, F., Krause, D., Deans, R., Keating, A., Prockop, D. & Horwitz, E. 2006. Minimal criteria for defining multipotent mesenchymal stromal cells: The International Society for Cellular Therapy position statement. Cytotherapy 8(4): 315-317.

Ellerström, C., Hyllner, J. & Strehl, R. 2010. Single cell enzymatic dissociation of human embryonic stem cells: A straightforward, robust, and standardized culture method. Human Embryonic Stem Cell Protocols 584: 121-134.

Golub, E.E. & Boesze-Battaglia, K. 2007. The role of alkaline phosphatase in mineralization. Current Opinion in Orthopaedics 18(5): 444-448.

Gronthos, S., Brahim, J., Li, W., Fisher, L., Cherman, N., Boyde, A., DenBesten, P., Robey, P.G. & Shi, S. 2002. Stem cell properties of human dental pulp stem cells. Journal of Dental Research 81(8): 531-535.

Hilkens, P., Gervois, P., Fanton, Y., Vanormelingen, J., Martens, W., Struys, T., Politis, C., Lambrichts, I. & Bronckaers, A. 2013. Effect of isolation methodology on stem cell properties and multilineage differentiation potential of human dental pulp stem cells. Cell and Tissue Research 353(1): 65-78.

Huang, G.T.J., Sonoyama, W., Chen, J. & Park, S.H. 2006. In vitro characterization of human dental pulp cells: Various isolation methods and culturing environments. Cell and Tissue Research 324(2): 225-236.

Huang, W., Carlsen, B., Rudkin, G., Berry, M., Ishida, K., Yamaguchi, D.T. & Miller, T.A. 2004. Osteopontin is a negative regulator of proliferation and differentiation in MC3T3-E1 pre-osteoblastic cells. Bone 34(5): 799-808.

Kermani, S., Megat Abdul Wahab, R., Zarina Zainol Abidin, I., Zainal Ariffin, Z., Senafi, S. & Hisham Zainal Ariffin, S. 2014. Differentiation capacity of mouse dental pulp stem cells into osteoblasts and osteoclasts. Cell Journal (Yakhteh) 16(1): 31-42.

Kruger, N.J. 2009. The Bradford method for protein quantitation. The Protein Protocols Handbook, edited by Walter, J.M. New Jersey: Humana Press. pp. 17-24.

Lopez-Cazaux, S., Bluteau, G., Magne, D., Lieubeau, B., Guicheux, J. & Alliot-Licht, B. 2006. Culture medium modulates the behaviour of human dental pulp-derived cells: technical note. Eur. Cell Mater. 11: 35-42.

Nadig, R.R. 2009. Stem cell therapy-hype or hope? A review.Journal of Conservative Dentistry 12(4): 131-138.

Nakashima, M. 1991. Establishment of primary cultures of pulp cells from bovine permanent incisors. Archives of Oral Biology 36(9): 655-663.

Nourbakhsh, N., Talebi, A., Mousavi, B., Nadali, F., Torabinejad, M., Karbalaie, K. & Baharvand, H. 2008. Isolation of mesenchymal stem cells from dental pulp of exfoliated human deciduous teeth. Cell J. 10(2): 101-108.

Raouf, A. & Seth, A. 2002. Discovery of osteoblast-associated genes using cDNA microarrays. Bone 30(3): 463-471.

Schnerch, A., Cerdan, C. & Bhatia, M. 2010. Distinguishing between mouse and human pluripotent stem cell regulation: The best laid plans of mice and men. Stem Cells 28(3): 419-430.

Seo, B.M., Miura, M., Gronthos, S., Bartold, P.M., Batouli, S.,Brahim, J., Young,  M., Robey, P.G.,Wang,  C.Y.  & Shi, S. 2004. Investigation of multipotent postnatal stem cells from human periodontal ligament. The Lancet 364(9429): 149-155.

Shi, S., Bartold, P., Miura, M., Seo, B., Robey, P. & Gronthos, S. 2005. The efficacy of mesenchymal stem cells to regenerate and repair dental structures. Orthodontics & Craniofacial Research 8(3): 191-199.

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. The Journal of Cell Biology 96(1): 191-198.

Thomas, C.H., Collier, J.H., Sfeir, C.S. & Healy, K.E. 2002. Engineering gene expression and protein synthesis by modulation of nuclear shape. Proceedings of the National Academy of Sciences 99(4): 1972-1977.

Yildirim, S. 2013. Dental pulp is a connective tissue. In Dental Pulp Stem Cells. New York: Springer. pp. 17-24.

*Corresponding author; email: drfarinawati@ukm.edu.my