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

  • Md Nazir Noorhidayah 
  • Ahmad Hafiz Zulkifly
  • Kamarul Ariffin Khalid
  • Ismail Zainol
  • Zaitunnatakhin Zamli
  • Munirah Sha’banEmail author
Original Article



This study aimed to observe the cartilaginous matrix production in SRY (sex determining region Y)-box 9 (SOX9)- and/or telomerase reverse transcriptase (TERT)-transfected chondrocytes from monolayer to three-dimensional (3D) culture.


The genes were transferred into chondrocytes at passage-1 (P1) via lipofection. The post-transfected chondrocytes (SOX9-, TERT- and SOX9/TERT) were analysed at P1, P2 and P3. The non-transfected group was used as control. The 3D culture was established using the chondrocytes seeded in a disc-shaped PLGA/fibrin and PLGA scaffolds. The resulting 3D “cells-scaffolds” constructs were analysed at week-1, -2 and -3. The histoarchitecture was evaluated using haematoxylin and eosin, alcian blue and safranin o stains. The quantitative sulphated glycosaminoglycan (sGAG) content was measured using biochemical assay. The cartilage-specific markers expression were analysed via real-time polymerase chain reaction.


All monolayer cultured chondrocytes showed flattened, fibroblast-like appearance throughout passages. Proteoglycan and sGAG were not detected at the pericellular matrix region of the chondrocytes. The sGAG content assay indicated the matrix production depletion in the culture. The cartilage-specific markers, COL2A1 and ACAN, were downregulated. However, the dedifferentiation marker, COL1A1 was upregulated. In 3D “cells-scaffolds” constructs, regardless of transfection groups, chondrocytes seeded in PLGA/fibrin showed a more uniform distribution and produced denser matrix than the PLGA group especially at week-3. Both sGAG and proteoglycan were clearly visualised in the constructs, supported by the increment of sGAG content, quantitatively. Both COL2A1 and ACAN were upregulated in SOX9/TERT-PLGA and SOX9/TERT-PLGA/fibrin respectively. While, COL1A1 was downregulated in SOX9/TERT-PLGA.


These findings indicated that the SOX9/TERT-transfected chondrocytes incorporation into 3D scaffolds facilitates the cartilage regeneration which is viable structurally and functionally.


Cartilage Chondrocytes Glycosaminoglycan Proteoglycan Tissue engineering 



The authors thanked the Kulliyyah of Allied Health Sciences (KAHS), IIUM, Kuantan Campus and Ministry of Science, Technology and Innovation (MOSTI) for providing Science Fund (SF14-012-0062). The authors also thanked Tissue Engineering and Regenerative Medicine Research Team, KAHS, IIUM, Kuantan Campus, Assoc. Prof. Dr. Tong Chuan He from University of Chicago, USA for pCDNA3-SOX9 and Dr. Bob Weinberg from Massachusetts Institute of Technology, USA for pBABE-neo-hTERT.

Compliance with ethical standards

Conflict of interest

The authors have declared that there is no conflict of interest.

Ethical statement

Animal ethical approval was granted by the Institutional Animal Care and Use Committee of International Islamic University Malaysia (IACUC-IIUM) (Reference No. IIUM/IACUC/Approval 2015/[5]/[24]).


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Copyright information

© The Korean Tissue Engineering and Regenerative Medicine Society 2019

Authors and Affiliations

  • Md Nazir Noorhidayah 
    • 1
  • Ahmad Hafiz Zulkifly
    • 2
  • Kamarul Ariffin Khalid
    • 2
  • Ismail Zainol
    • 3
  • Zaitunnatakhin Zamli
    • 1
  • Munirah Sha’ban
    • 4
    Email author
  1. 1.Department of Biomedical Science, Kulliyyah of Allied Health SciencesInternational Islamic University Malaysia (IIUM)KuantanMalaysia
  2. 2.Department of Orthopaedics, Traumatology and Rehabilitation, Kulliyyah of MedicineInternational Islamic University Malaysia (IIUM)KuantanMalaysia
  3. 3.Department of Chemistry, Faculty of Science and MathematicsUniversiti Pendidikan Sultan Idris (UPSI)Tanjong MalimMalaysia
  4. 4.Department of Physical Rehabilitation Sciences, Kulliyyah of Allied Health SciencesInternational Islamic University Malaysia (IIUM)KuantanMalaysia

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