Sains Malaysiana 48(8)(2019): 1697–1706

http://dx.doi.org/10.17576/jsm-2019-4808-15

An in vitro Three-Dimensional Co-Culture System for Ameloblastoma Modelling

(Sistem Ko-Kultur Tiga Dimensi secara in vitro untuk Pemodelan Ameloblastoma)

SOO LENG LEE¹*, ZAINAL ARIFF ABDUL RAHMAN¹, HIDETSUGU TSUJIGIWA², MEI HAMADA³, KIYOFUMI TAKABATAKE³, KEISUKE NAKANO³, HITOSHI NAGATSUKA³ & CHONG HUAT SIAR¹

¹Department of Oral and Maxillofacial Clinical Sciences, Faculty of Dentistry, University of Malaya, 50603 Kuala Lumpur, Federal Territory, Malaysia

²Laboratory of Histopathology, Department of Life Science, Faculty of Science, Okayama University of Science, Okayama, Japan

³Department of Oral Pathology and Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan

Diserahkan: 18 September 2018/Diterima: 16 Mei 2019

ABSTRACT

Ameloblastoma, the most clinically significant odontogenic epithelial tumor, is a locally-invasive and destructive lesion in the jawbones. However, the nature of this infiltrativeness and destructive behavior remains ill-understood. To address this, we established an in vitro three-dimensional (3D) co-culture system to simulate an amelobastoma disease model aimed at investigating the interactions between tumor cells and osteoblasts. Osteoblastic cell lines (KUSA/A1 and MC3T3-E1) and one stromal cell line (ST2) were separately co-seeded with ameloblastoma-derived cell line (AM-1) in a collagen scaffold (representing the extracellular bone matrix) and incubated with mineralization medium. Immunohistochemistry, double immunofluorescence and mineralization assay were performed. Only AM-1/KUSA-A1 co-culture showed a significant increase in AM-1 cell count, suggesting that heterotypic cell-cell interaction promotes tumoral cell growth, while formation of visible AM-1 epithelial nest-like structures resembling ameloblastoma cells in their native state, suggest morphodifferentiation. A RANK-high, RANKL-low and osteoprotegerin-low immunoprofile in co-culture AM-1 cells implies deregulated osteoclastogenesis. Mineralization assays showed diminished calcification in AM-1/KUSA-A1 co-culture extracellular matrix suggesting an altered local bone metabolism. In contrast, KUSA/A1 monocultures showed abundant extracellular matrix calcification. Taken together, these results suggest that a 3D co-culture system as an amelobastoma disease model provides insights that bidirectional ameloblastoma-osteoblastic interactions might play a role in modulating tumor growth and osteoclastogenesis.

Keywords: Ameloblast; ameloblastoma modelling; co-culture system; pre-osteoblast

ABSTRAK

Ameloblastoma, tumor epitelium odontogenik yang paling umum secara klinikal, ialah lesi invasif setempat dan lesi memusnah yang didapati di tulang rahang. Walau bagaimanapun, sifat penyusupan dan perilaku yang merosakkan masih tidak difahami. Untuk menangani ini, kami menubuhkan satu sistem in vitro tiga dimensi (3D) untuk mensimulasikan model penyakit amelobastoma, bertujuan untuk mengkaji interaksi antara tumor dan sel osteoblastik. Sel-sel osteoblastik (KUSA/A1 dan MC3T3-E1) dan satu sel stromal (ST2) bersandarkan dengan sel-sel tumor ameloblastoma, AM-1 secara berasingan dalam gel kolagen (mewakili matriks tulang ekstrasel) dan diinkubasi dengan medium pemineralan. Imunohistokimia, imunopendarfluor ganda dua dan asai mineral dijalankan. Hanya kultivar AM-1/KUSA-A1 menunjukkan kenaikan ketara dalam jumlah sel AM-1, menunjukkan bahawa interaksi sel heterotip menggalakkan pertumbuhan sel tumor, manakala pembentukan struktur seperti sarang epitelium AM-1 kelihatan sebagai sel ameloblastoma dalam keadaan asalnya, mencadangkan pembezaan morfotip. RANK-Tinggi, RANKL-rendah dan osteoprotegerin-rendah imunoprofil dalam ko-kultur dengan sel AM-1 mencadangkan osteoklastogenesis yang tidak terkawal. Pemineralan asai juga menunjukkan bahawa kalsifikasi dalam matriks ekstrasel AM-1/KUSA-A1 telah mencadangkan perubahan dalam metabolisme tulang setempat. Sebaliknya, monokultur KUSA/A1 menunjukkan kalsifikasi matriks ekstrasel yang signifikan. Secara eksplisit, keputusan ini menunjukkan bahawa sistem ko-kultur 3D sebagai model penyakit amelobastoma memberikan pandangan terperinci terhadap interaksi antara ameloblastoma dan osteoblas dan keupayaannya untuk memainkan peranan penting dalam pengubahan pertumbuhan tumor dan osteoklastogenesis.

Kata kunci: Ameloblas; pemodelan ameloblastoma; pra-osteoblas; sistem ko-kultur

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*Pengarang untuk surat-menyurat; email: leng527@siswa.um.edu.my