Sains Malaysiana 49(5)(2020): 1067-1080

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 menggunakanSry (Penentu Jantina RantauY)-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

Received: 29 July 2019/Accepted: 29 January 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

REFERENCES

Alsberg, E., Anderson, K.W., Albeiruti, A., Rowley, J.A. & Mooney, D.J. 2002. Engineering growing tissues. Proceedings of the National Academy of Sciences 99(19): 12025-12030.

Calabrese, G., Gulino, R., Giuffrida, R., Forte, S., Figallo, E., Fabbi, C., Salvatorelli, L., Memeo, L., Gulisano, M. & Parenti, R. 2017. In vivo evaluation of biocompatibility and chondrogenic potential of a cell-free collagen-based scaffold. Frontiers in Physiology 8: 984.

Choi Jiye & Yong Jeong 2017. Elevated emotional contagion in a mouse model of Alzheimer’s Disease is associated with increased synchronization in the insula and amygdala. Scientific Reports 7: 1-9.

Deng, Y.H., Lei, G.H., Lin, Z.X., Yang, Y.H.,  Lin, H. & Tuan, R.S. 2019. Engineering hyaline cartilage from mesenchymal stem cells with low hypertrophy potential via modulation of culture conditions and Wnt/β-catenin pathway. Biomaterials 192: 569-578.

Elakkiya Venugopal, Narmadha Rajeswaran, K. Santosh Sahanand, Amitava Bhattacharyya & Selvakumar Rajendran. 2019. In vitro evaluation of phytochemical loaded electrospun gelatin nanofibers for application in bone and cartilage tissue engineering. Biomedical Materials 14(1): 015004.

Inada, M., Wang, Y.M., Byrne, M.H., Rahman, M.U., Miyaura, C., Lopez-Otin, C. & Krane, S.M. 2004. Critical roles for collagenase-3 (Mmp13) in development of growth plate cartilage and in endochondral ossification. Proceedings of the National Academy of Sciences 101(49): 17192-17197.

Karlsson, M. & Björnsson, S. 2001. Quantitation of proteoglycans in biological fluids using Alcian Blue. In Methods in Molecular Biology, edited by Lozzo, R.V. Totowa, NJ: Prote. Humana Press Inc.

Kavand, H., Van Lintel, H. & Renaud, P. 2019. Efficacy of pulsed electromagnetic fields and electromagnetic fields tuned to the ion cyclotron resonance frequency of Ca2+ on chondrogenic differentiation. Journal of Tissue Engineering and Regenerative Medicine 13(5): 799-811.

Kudva, A.K., Luyten, F.P. & Patterson, J. 2018. In vitro screening of molecularly engineered polyethylene glycol hydrogels for cartilage tissue engineering using periosteum-derived and ATDC5 cells. International Journal of Molecular Sciences 19(11): 3341.

Joydip Kundu, Shim, J.H., Jang, J.N., Kim, S.W.  & Cho, D.W. 2013. An additive manufacturing-based PCL-Alginate- chondrocyte bioprinted scaffold for cartilage tissue engineering. Journal of Tissue Engineering and Regenerative Medicine 9(11): 1286-1297.

Lih, E., Kum, C.H., Park, W.R., Chun, S.Y., Cho, Y.J., Joung, Y.K. & Park, K.S. 2018. Modified magnesium hydroxide nanoparticles inhibit the inflammatory response to biodegradable poly(lactide- co-glycolide) implants. ACS Nano. 12(7): 6917-6925.

Liu, Y., Tian, K., Hao, J., Yang, T.,  Geng, X.L. & Zhang, W.G. 2019. Biomimetic poly (glycerol sebacate)/polycaprolactone blend scaffolds for cartilage tissue engineering. Journal of Materials Science: Materials in Medicine 30(5): 53.

Mohd Yunus Mohd Heikal, Shuid Ahmad Nazrun, Kien Hui Chua & Abd Ghafar Norzana. 2019. Stichopus chloronotus aqueous extract as a chondroprotective agent for human chondrocytes isolated from osteoarthitis articular cartilage in vitro. Cytotechnology 71(2): 521-537.

Munirah Sha’ban, Ruszymah, Hj Idrus, Samsudin Osman Cassim, Badrul, A.H.M.Y., Azmi Baharudin & Aminuddin Saim. 2008a. Autologous versus pooled human serum for articular chondrocyte growth. Journal of Orthopaedic Surgery 16(2): 220-229.

Munirah Sha’ban, Soon Hee Kim., Ruszymah Hj Idrus & Gilson Khang. 2008b. The use of fibrin and poly(lactic-co-glycolic acid) hybrid scaffold for articular cartilage tissue engineering: An in vivo analysis. European Cells and Materials 15: 41-52.

Noorhidayah Md Nazir, Ahmad Hafiz Zulkifly, Kamarul Ariffin Khalid, Ismail Zainol, Zaitunnatakhin Zamli & Munirah Sha'ban. 2019. Matrix production in chondrocytes transfected with sex determining region Y-Box 9 and telomerase reverse transcriptase genes: An in vitro evaluation from monolayer culture to three-dimensional culture. Tissue Engineering and Regenerative Medicine 16(3): 285-299.

Ogura, T., Bryant, T., Merkely, G. & Minas, T. 2019. Autologous chondrocyte implantation for bipolar chondral lesions in the patellofemoral compartment: Clinical outcomes at a mean 9 years’ follow-up. American Journal of Sports Medicine 47(4): 837-846.

Prosser, A., Scotchford, C., Roberts, G., Grant, D. & Sottile, V.  2019. Integrated multi-assay culture model for stem cell chondrogenic differentiation. International Journal of Molecular Sciences 20(4): 951.

Rozlin Abdul Rahman, Norhamiza Mohamad Sukri, Noorhidayah Md Nazir, Muhammad Aa'zamuddin Ahmad Radzi, Ahmad Hafiz Zulkifly, Aminudin Che Ahmad, Abdurezak Abdulahi Hashi, Suzanah Abdul Rahman & Munirah Sha'ban. 2015. The potential of 3-dimensional construct engineered from poly(lactic-co-glycolic acid)/fibrin hybrid scaffold seeded with bone marrow mesenchymal stem cells for in vitro cartilage tissue engineering. Tissue and Cell 47(4): 420-430.

Sabatino, M.A., Santoro, R., Gueven, S., Jaquiery, C., Wendt, D.J., Martin, I., Moretti, M. & Barbero, A. 2012. Cartilage graft engineering by co-culturing primary human articular chondrocytes with human bone marrow stromal cells. Pediatric Endocrinology Reviews 9(12): 1394-1403.

Sasano, Y., Furusawa, M., Ohtani, H., Mizoguchi, I., Takahashi, I. & Kagayama, M. 1996. Chondrocytes synthesize type I collagen and accumulate the protein in the matrix during development of rat tibial articular cartilage. Anatomy and Embryology 194(3): 247-252.

Schmitz, N., Laverty, S., Kraus, V.B. & Aigner, T. 2010. Basic methods in histopathology of joint tissues. Osteoarthritis and Cartilage 18: 113-116.

Vermeulen, S., Vasilevich, A., Tsiapalis, D., Roumans, N., Vroemen, P., Beijer, N.R.M., Dede-Eren, A., Zeugolis, D. & de Boer, J. 2019. Identification of topographical architectures supporting the phenotype of rat tenocytes. Acta Biomaterialia 83: 277-290.

Wang, T.Y., Lai, J.H., Han, L-H., Tong, X.M. & Yang, F. 2016. Modulating stem cell-Chondrocyte interactions for cartilage repair using combinatorial extracellular matrix-containing hydrogels. Journal of Materials Chemistry B 4(47): 7641-7650.

Yamamoto, K.,  Okano, H., Miyagawa, W., Visse, R., Shitomi, Y., Santamaria, S., Dudhia, J., Troeberg, L., Strickland, D.K., Hirohata, S. & Nagase, H. 2016. MMP-13 is constitutively produced in human chondrocytes and co-endocytosed with ADAMTS-5 and TIMP-3 by the endocytic receptor LRP1. Matrix Biology 56: 57-73.

Zhang, X.D., Qi, L., Chen, Y.H., Xiong, Z.Z., Li, J.J., Xu, P., Pan, Z.Q., Zhang, H.Z., Chen, Z.X., Xue, K. & Liu, K. 2019. The in vivo chondrogenesis of cartilage stem/progenitor cells from auricular cartilage and the perichondrium. American Journal of Translational Research 11(5): 2855-2865.

Zhang, X.W., Wu, S.L., Naccarato, T., Prakash-Damani, M., Chou, Y., Chu, C-Q. & Zhu, Y. 2017. Regeneration of hyaline-like cartilage in situ with SOX9 stimulation of bone marrow-derived mesenchymal stem cells. PLoS ONE 12(6): e0180138.

Zhu, Y.Q., Zhang, Y.Y., Yu, L., Tao, R., Xia, H.T., Zheng, R., Shi, Y., Tang, S.J., W.J., Zhang., Liu, W., Cao, Y.L. & Zhou, G.D. 2014. Influence of Chm-I knockout on ectopic cartilage regeneration and homeostasis maintenance. Tissue Engineering Part A 21(3-4): 782-792.

*Corresponding author; email: munirahshaban@iium.edu.my