Sains Malaysiana 40(4)(2011): 305–309

Proliferation and Biochemical Analyses of Osteoblast/Osteoclast Differentiation from Human Mononucleated Cells

(Analisis Proliferasi dan Biokimia Terhadap Sel Mononukleus Manusia yang dibezakan kepada Sel Osteoblas/Osteoklas)

Siti Afeefah Mohamad Yusof¹, Rohaya Megat Abdul Wahab², Sahidan Senafi¹, Zaidah Zainal Ariffin³, MohamadAbdul Razak⁴ & Shahrul HishamZainal Ariffin¹ *

¹Pusat Pengajian Biosains & Bioteknologi, Fakulti Sains dan Teknologi

Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia

²Jabatan Ortodontik, Fakulti Pergigian, Universiti Kebangsaan Malaysia

Jalan Raja Muda Aziz, 50300 Kuala Lumpur, Malaysia

³Jabatan Mikrobiologi, Fakulti Sains Gunaan, Universiti Teknologi MARA

40450, Shah Alam, Selangor, Malaysia

⁴Jabatan Ortopedik, Pusat Perubatan, Universiti Kebangsaan Malaysia

Jalan Yaacob Latif, Bandar Tun Razak, 56000 Kuala Lumpur, Malaysia

Diserahkan: 9 Disember 2009 / Diterima: 21 Oktober 2010

ABSTRACT

Stem cell is defined as the ability of the cell to proliferate themselves and differentiate into more than one type of cells. Human mononucleated cell (MN cell) is a suspension cell that was isolated from peripheral blood that was originated from monocyte-machrophage lineage or hematopoietic stem cells. The cells were cultured for 30 days in complete media (CM) which consist of Alpha Minimal Essential Medium (αMEM) with 2% (v/v) Penicillin-Streptomycin and 10% (v/v) Newborn Calf Serum (NBCS). The respective cells were differentiated at day 7 after in vitro proliferation in CM into osteoblastic cells by adding ascorbic acid and β-glycerophosphate. In addition, human recombinant Receptor Activator of Nuclear Factor-β Ligand (hrRANKL) and human recombinant Macrophage-Colony Stimulating Factor (hrM-CSF) were added to induce osteoclastic differentiation of MN cells. Cells that were cultured in CM served as a control and were subjected to the same approach as differentiated cells. The 30 days cultured cells in CM showed a significant increment (p < 0.05) of viable cells compared to day 0 (n=3). The specific activity of Alkaline Phosphatase (ALP) for osteoblast differentiation and Tartrate Resistant Acid Phosphatase (TRAP) for osteoclast differentiation were evaluated via biochemical assay until day 14 and day 10 for osteoblast and osteoclast sample, respectively. ALP and TRAP enzyme showed a significant increment (p < 0.05) after 14 and 10 days of differentiation compared to control cells. As a conclusion, human mononucleated cells are believed to have the potential to be defined as a multipotent stem cell based on their fulfillment of stem cell characteristics.

Keywords: Enzymological analysis; mononucleated; Osteoblast; Osteoclast

ABSTRAK

Sel stem ialah sel yang mempunyai keupayaan mengganda dan membeza kepada lebih daripada satu jenis sel. Sel mononukleus (MN) manusia merupakan sel terampai yang diasingkan daripada sel darah periferi; berasal daripada turunan monosit-makrofaj iaitu merupakan sel stem hematopoitik. Sel tersebut dikultur selama 30 hari di dalam media lengkap (CM) yang mengandungi ‘Alpha Minimal Essential Medium’ (αMEM), 2% (v/v) Penisilin-Streptomicin and 10% (v/v) ‘Newborn Calf Serum’ (NBCS). Sel mononukleus dibezakan pada hari ketujuh pengkulturan in vitro kepada sel osteoblas dengan menambahkan asid askorbik dan β-gliserofosfat. Bagi mengaruhkan pembezaan MN kepada sel osteoklas, ‘Receptor Activator of Nuclear Factor-β Ligand’ rekombinan manusia (hrRANKL) dan ‘Macrophage-Colony Stimulating Factor’ rekombinan manusia (hrM-CSF) ditambahkan ke dalam CM. Sel yang dikultur di dalam CM bertindak sebagai kawalan. Sel yang dikultur selama 30 hari di dalam CM menunjukkan peningkatan sel viabel yang signifikan (p < 0.05) berbanding hari 0 (n=3). Analisis biokimia dilakukan terhadap enzim Alkalin Fosfatase (ALP) bagi sel osteoblas sehingga hari 14 pembezaan dan enzim Asid Fosfatase Rintang Tartrat (TRAP) bagi sel osteoklas sehingga hari 10 pembezaan. Enzim ALP dan TRAP pada sel osteoblas dan sel osteoklas masing-masing menunjukkan peningkatan yang signifikan (p < 0.05) pada hari 14 dan 10 berbanding sel kawalan. Sebagai kesimpulan, sel MN manusia dipercayai mempunyai potensi untuk dikelaskan sebagai sel stem multipoten kerana memenuhi ciri-ciri sel stem yang telah disebutkan.

Kata kunci: Analisis enzimologi; mononukleus; Osteoblas; Osteoklas

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*Pengarang untuk surat-menyurat; email:  hisham@ukm.my