Sains Malaysiana 49(5)(2020): 1067-1088

http://dx.doi.org/10.17576/jsm-2020-4905-11

The Cartilaginous Tissue Formation using Sry (Sex Determining Region Y)-BOX9 and Telomerase Reverse Transcriptase Genes Transfected Chondrocytes: In vivo Approach

(Pembentukan Tisu Tulang Rawan menggunakan Sry (Penentu Jantina Rantau Y)-BOX9 dan Telomerase Gen Transkripsi Berbalik Kondrosit Transfeksi: Pendekatan in vivo)

NOORHIDAYAH MD NAZIR¹, AHMAD HAFIZ ZULKIFLY², KAMARUL ARIFFIN KHALID², ISMAIL ZAINOL³, ZAITUNNATAKHIN ZAMLI¹ & MUNIRAH SHA’BAN⁴*

¹Department of Biomedical Science, Kulliyyah of Allied Health Sciences, International Islamic University Malaysia (IIUM), Jalan Sultan Ahmad Shah, Bandar Indera Mahkota, 25200 Kuantan, Pahang Darul Makmur, Malaysia

²Department of Orthopaedics, Traumatology and Rehabilitation, Kulliyyah of Medicine, International Islamic University Malaysia (IIUM), Jalan Hospital Campus, 25100 Kuantan, Pahang Darul Makmur, Malaysia

³Department of Chemistry, Faculty of Science and Mathematics, Universiti Pendidikan Sultan Idris (UPSI), 35900 Tanjong Malim, Perak Darul Ridzuan, Malaysia

⁴Department of Physical Rehabilitation Sciences, Kulliyyah of Allied Health Sciences,

International Islamic University Malaysia (IIUM), Jalan Sultan Ahmad Shah, Bandar Indera Mahkota, 25200 Kuantan, Pahang Darul Makmur, Malaysia

Diserahkan: 29 Julai 2019/Diterima: 29 Januari 2020

ABSTRACT

The shortage of organ supply reduces the success rate of organ transplantation. Hence, tissue regeneration has been initiated with the intention of improving the available treatment modalities. Articular cartilage is a suitable tissue for this purpose due to its limited self-heal ability. This study aims to evaluate the cartilaginous properties of in vivo constructs formed using chondrocytes transfected with the combination of sry (sex determining region y)-box9 (SOX9) and telomerase reverse transcriptase (TERT) genes (SOX9/TERT-transfected chondrocytes) seeded on a three-dimensional (3D) poly(lactic-co-glycolic) acid (PLGA)-based scaffold. The rabbit’s articular chondrocytes (n=6) were transfected with SOX9 and TERT genes via lipofection. The non-transfected chondrocyte (NTC) was used as a control. A total of 1×10⁵ cells were seeded on a PLGA and PLGA/fibrin hybrid scaffolds to form constructs. The resulted constructs were SOX9/TERT-PLGA/fibrin, NTC-PLGA/fibrin, SOX9/TERT-PLGA and, NTC-PLGA. All constructs were cultured for three weeks prior to subcutaneous implantation into the athymic mice for two and four weeks. The constructs’ structural and functional aspects were evaluated using macroscopic observation, compression-stress analysis, histology, quantitative sulphated glycosaminoglycan (sGAG) assay and cartilage-specific genes (ACAN, COL2A1, SOX9), TERT, and MMP13 expression analysis. The constructs demonstrated a cartilage-like appearance. The constructs’ rigidity corresponded to the homogenous cells and extracellular matrix distribution in the week-4 constructs. Correspondingly, the cartilaginous matrix components were visualised at the pericellular matrix region of the construct, supported by the increment of quantitative sGAG content. The SOX9/TERT-PLGA/fibrin exhibited better genes expression and cartilaginous phenotypes than the other construct groups. The SOX9/TERT-PLGA/fibrin construct facilitated cartilaginous tissue formation.

Keywords: Cartilage; chondrocytes; in vivo; SOX9; TERT; transfection

ABSTRAK

Kekurangan bekalan organ menurunkan kadar kejayaan pemindahan organ. Oleh itu, penjanaan semula tisu telah diusahakan bagi memperbaiki modaliti rawatan sedia ada. Rawan artikul ialah tisu yang sesuai untuk kegunaan ini oleh sebab kebolehan swapulihnya yang terbatas. Kajian ini bertujuan untuk menilai ciri-ciri tulang rawan binaan in vivo yang dibentuk melalui transfeksi gabungan gen sry (penentu jantina rantau y)-box9 (SOX9) dan gen telomerase transkriptase membalik(TERT) ke dalam sel kondrosit yang disemai atas kerangka tiga dimensi (3D) berasaskan asidpoli(laktik-ko-glikolik) (PLGA). Sel kondrosit artikul arnab (n=6) telah ditransfeksi dengan gen SOX9 dan TERT melalui kaedah lipofeksi. Sel kondrosit tanpa transfeksi (NTC) telah digunakan sebagai kawalan. Sejumlah 1×10⁵ sel telah disemai atas kerangka PLGA 3D dan kerangka hibrid PLGA/fibrin untuk membentuk binaan. binaan yang terhasil ialahSOX9/TERT-PLGA/fibrin, NTC-PLGA/fibrin, SOX9/TERT-PLGA dan NTC-PLGA. Semua binaan telah dikultur selama tiga minggu sebelum diimplantasi secara subkutaneus ke dalam tikus tanpa timus selama dua dan empat minggu. Aspek struktur dan fungsian binaan telah dinilai menggunakan pemerhatian makroskopik, analisis tegasan mampatan, histologi, asai kuantitatif glikosaminoglikan sulfat(sGAG) dananalisis ekspresi gen khusus tulang rawan (ACAN, COL2A1, SOX9), TERT danMMP13. Binaan menunjukkan rupa tulang rawan. Ketegaran binaan sejajar dengan taburan sel homogen dan matriks ekstrasel pada binaan minggu ke-4. Komponen matriks tulang rawan telah dilihat pada binaan di bahagian matriks periselular, disokong oleh kenaikan kandungan kuantitatif sGAG. SOX9/TERT-PLGA/fibrin mempamerkan ekpresi gen dan fenotip tulang rawan yang lebih baik berbanding kumpulan binaan yang lain. Binaan SOX9/TERT-PLGA/fibrin memudahkan pembentukan tisu tulang rawan.

Kata kunci: In vivo; sel kondrosit; SOX9; TERT; transfeksi; tulang rawan

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*Pengarang untuk surat-menyurat; email: munirahshaban@iium.edu.my