The Re-Expression of Collagen Type 2, Aggrecan and Sox 9 in Tissue-Engineered Human Articular Cartilage

( Munirah Sha`ban ),( Aminuddin Bin Saim ),( Samsudin Osman Cassim ),( Chua Kien Hui ),( Fuzina Nor Hussein ),( Ruszymah Bt Hj Idrus ) 한국조직공학과 재생의학회 2005 조직공학과 재생의학 Vol.2 No.4

This study was designed to verify the optimal · basic culture media that promote chondrocytes proliferation in vitro in order to facilitate adequate amount of chondrocytes for cartilage reconstructionas well as maintaining cartilage specific phenotype. Human articular chondrocytes were cultured in three types of basic culture media Ham`s F12, DMEM and the equivalent mixture of F12:DMEM. Cultured chondrocytes were trypsinized as they reached confluency. The viability and total number of cell were recorded at every passage. Large-scale culture expansion was used to reconstruct tissue-engineered cartilage. Quantitative RT-PCR analysis was used to evaluate the expression of collagen Type II, collagen Type I, aggrecan and Sox 9 gene, both in monolayer culture and in the engineered cartilage. The mixture of F12:DMEM promotes significantly greater (p<0.05) chondrocytes proliferation at every passage compared to the individual medium. Monolayer cultured chondrocytes exhibited down-regulation expression pattern of collagen Type II gene, aggrecan and Sox 9, whilst the expression of collagen Type I is up-regulated. Tissue-engineered cartilage morphologically and histologically resembled normal hyaline cartilage. Moreover, tissue-engineered cartilage re-expressed the specific chondrogenesis markers; collagen Type II, aggrecan and Sox 9. In conclusion, the mixture of F12:DMEM enhanced human articular chondrocytes proliferation thus provided adequate amount of chondrocytes for cartilage reconstruction. The new cartilage formed phenotypically resembles native cartilage. This results hold promise for the use of tissue-engineered cartilage implant for future orthopaedic reconstructive surgery.

Sox-9 Transient Transfection Enhances Chondrogenic Expression of Osteoarthritic Human Articular Chondrocytes In Vitro: Preliminary Analysis

( Munirah Sha` Ban ),( Samsudin Osman Cassim ),( Nor Hamdan Mohd Yahya ),( Aminuddin Bin Saim ),( Ruszymah Bt Hj Idrus ) 한국조직공학·재생의학회 2011 조직공학과 재생의학 Vol.8 No.1

In this study, we are taking step to actively manage osteoarthritis that may help gain control over osteoar-thritic 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.

Tissue Engineering of Articular Cartilage: From Bench to Bed-side

Rozlin Abdul Rahman,Muhammad Aa’zamuddin Ahmad Radzi,Norhamiza Mohamad Sukri,Noorhidayah Md Nazir,Munirah Sha’ban 한국조직공학과 재생의학회 2015 조직공학과 재생의학 Vol.12 No.1

Degeneration or defect of articular cartilage is a major predicament and if it is left untreated, it may leadto progressive damage and disability affecting every one disregard of their age. Although nonsurgical managementof articular cartilage injury has remained largely the same over many years, surgical treatment keeps on evolving. Restorative techniques, mainly the cell-based therapies and autologous or allograft transplants continue to expand,giving surgeons more options for biologic reconstruction of the articular surfaces. Hence the field of articular cartilagetissue engineering which seeks to repair, restore and improve injured or diseased articular cartilage functionalityhas aroused deep interest and holds great potential for improving articular cartilage therapy. However despite thisgreat evolution, therapeutic uncertainty in the restoration of damaged cartilage using tissue engineering approachesstill remains unclear for the surgeon treating patients to make evidence-based decisions. This paper will give a generalidea to different level of audiences in understanding the concept of tissue engineering from bench to bed-sideregarding recent developments in this exciting field.

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

Noorhidayah Md Nazir,Ahmad Hafiz Zulkifly,Kamarul Ariffin Khalid,Ismail Zainol,Zaitunnatakhin Zamli,Munirah Sha’ban 한국조직공학과 재생의학회 2019 조직공학과 재생의학 Vol.16 No.3

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