Sox-9 transient transfection enhances chondrogenic expression of osteoarthritic human articular chondrocytes in vitro: Preliminary analysis

Munirah Sha'ban, Samsudin Osman Cassim, Nor Hamdan Mohamad Yahaya, Aminuddin Bin Saim, Ruszymah Idrus

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

In this study, we are taking step to actively manage osteoarthritis that may help gain control over osteoarthritic pain and delay the degenerative changes in articular cartilage in future. We transiently over expressed cartilage transcriptional factor, human sox-9 gene in chondrocytes derived from consented osteoarthritic patients after joint surgery. The expression vector carrying human sox-9 gene, pAdTrack-sox9 was transformed into One Shot® TOP10 Chemically Competent E. coli according to the manufacturer protocol. Plasmid purification was performed in accordance with QIAGEN® plasmid purification kit procedure. We compared the efficiency between two transfection techniques i.e. lipofection using Lipofectamine- 2000 kit from Invitrogen, USA and nucleofection using Human Chondrocytes Nucleofector® kit from Amaxa Biosystem, Germany. Chondrocytes were cultured and transfected with sox-9 gene at passage 1 according to the manufacturers' protocols. Transfected chondrocytes were expanded until passage 3. Expression of chondrogenic markers namely collagen type II, aggrecan core protein and sox-9 were evaluated by quantitative RT-PCR method using iScriptTM One Step RT-PCR Kit with SYBR® Green, BIO-RAD. Chondrogenic dedifferentiation marker, collagen type I was also analyzed using the quantitative RT-PCR method. Expression level of each targeted gene was normalized to the housekeeping gene, human glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Overall efficiency ranging from 50% to 60% could be achieved using both transfection techniques. Transiently transfecting cells demonstrated remarkable competency sustaining specific chondrogenic genes namely collagen type II, aggrecan core protein and sox-9, significantly better than in the non-transfected cells. It is believed that this preliminary finding has to be extended to develop its full potential since sox-9 transcription factor is essential for chondrocyte differentiation and cartilage formation. Sox9 gene therapy would delay the degenerative changes in articular cartilage which is consistent to the up-regulation of cartilage-specific markers especially collagen type II synthesis in vivo.

Original languageEnglish
Pages (from-to)32-41
Number of pages10
JournalTissue Engineering and Regenerative Medicine
Volume8
Issue number1
Publication statusPublished - Jan 2011

Fingerprint

Chondrocytes
Cartilage
Transfection
Genes
Joints
Collagen Type II
Collagen
Aggrecans
Articular Cartilage
Polymerase Chain Reaction
Plasmids
Purification
Glyceraldehyde-3-Phosphate Dehydrogenases
Essential Genes
Proteins
Gene therapy
Collagen Type I
Transcription factors
Osteoarthritis
Genetic Therapy

Keywords

  • Articular chondrocytes
  • Lipofection
  • Nucleofection
  • Osteoarthritis
  • Sox-9 transfection

ASJC Scopus subject areas

  • Medicine (miscellaneous)
  • Biomedical Engineering

Cite this

Sox-9 transient transfection enhances chondrogenic expression of osteoarthritic human articular chondrocytes in vitro : Preliminary analysis. / Sha'ban, Munirah; Cassim, Samsudin Osman; Mohamad Yahaya, Nor Hamdan; Bin Saim, Aminuddin; Idrus, Ruszymah.

In: Tissue Engineering and Regenerative Medicine, Vol. 8, No. 1, 01.2011, p. 32-41.

Research output: Contribution to journalArticle

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