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Phenotypic expression of collagen type II and collagen type I gene in monolayer culture of human auricular chondrocytes

Nur Adelina AN, Aminuddin BS, Munirah S, Chua KH, Fuzina NH, Saim L, et al.

Med J Malaysia, 2004 May;59 Suppl B:188-9.
PMID: 15468881

Cartilage is regularly needed for reconstructive surgery. Basic research in tissue engineering is necessary to develop its full potential. We presented here the expression profile of type II collagen gene and type I collagen gene in human auricular monolayer culture expansion. Cultured chondrocytes documented a reduction in the expression level of collagen type II gene whilst collagen type I gene was gradually expressed through all the passages. This study demonstrated that human auricular chondrocytes lose its phenotypic expression during monolayer culture expansion. Further studies are required to enhance cartilage specific gene expression, collagen type II throughout the in vitro culture.

Matched MeSH terms: Collagen Type II/genetics*
Interaction between insulin-like growth factor-1 with other growth factors in serum depleted culture medium for human cartilage engineering

Chua KH, Aminuddin BS, Fuzina NH, Ruszymah BH

Med J Malaysia, 2004 May;59 Suppl B:7-8.
PMID: 15468792

The regulation roles of insulin-like growth factor-1 (IGF-1) with basic fibroblast growth factor (bFGF) and transforming growth factor beta 2 (TGFbeta2) in human nasal septum chondrocytes monolayer culture and cartilage engineering was investigated in this study. The role of IGF-1 with bFGF and TGFbeta2 was investigated by measuring chondrocyte growth kinetic and collagen genes expression. IGF-1 together with bFGF and TGFbeta2 promote cartilage tissue engineering, increase type II collagen expression and enhance the histological features of engineered cartilage.

Matched MeSH terms: Collagen Type II/genetics
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 N, Zulkifly AH, Khalid KA, Zainol I, Zamli Z, Sha'ban M

Tissue Eng Regen Med, 2019 06;16(3):285-299.
PMID: 31205857 DOI: 10.1007/s13770-019-00191-1

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.

Matched MeSH terms: Collagen Type II/genetics
Fulltext A novel de novo mutation in COL2A1 gene associated with fetal skeletal dysplasia

Chu FC, Hii LY, Hung TH, Lo LM, Hsieh TT, Shaw SW

Taiwan J Obstet Gynecol, 2021 Mar;60(2):359-362.
PMID: 33678343 DOI: 10.1016/j.tjog.2021.01.017

OBJECTIVE: Skeletal dysplasias, caused by genetic mutations, are a heterogenous group of heritable disorders affecting bone development during fetal life. Stickler syndrome, one of the skeletal dysplasias, is an autosomal dominant connective tissue disorder caused by abnormal collagen synthesis owing to a genetic mutation in COL2A1.
CASE REPORT: We present the case of a 38-year-old multipara woman whose first trimester screening showed a normal karyotype. However, the bilateral femur and humerus length symmetrically shortened after 20 weeks. Next-generation sequencing for mutations in potential genes leading to skeletal dysplasia detected a novel de novo mutation (c.1438G > A, p.Gly480Arg) in COL2A1, causing Stickler syndrome type 1. This pathogenic mutation might impair or destabilize the collagen structure, leading to collagen type II, IX, and XI dysfunction.
CONCLUSION: We identified a novel de novo mutation in COL2A1 related to the STL1 syndrome and delineated the extent of the skeletal dysplasia disease spectrum.

Matched MeSH terms: Collagen Type II/genetics*
Fibrin promotes proliferation and matrix production of intervertebral disc cells cultured in three-dimensional poly(lactic-co-glycolic acid) scaffold

Sha'ban M, Yoon SJ, Ko YK, Ha HJ, Kim SH, So JW, et al.

J Biomater Sci Polym Ed, 2008;19(9):1219-37.
PMID: 18727862 DOI: 10.1163/156856208785540163

Previously, we have proven that fibrin and poly(lactic-co-glycolic acid) (PLGA) scaffolds facilitate cell proliferation, matrix production and early chondrogenesis of rabbit articular chondrocytes in in vitro and in vivo experiments. In this study, we evaluated the potential of fibrin/PLGA scaffold for intervertebral disc (IVD) tissue engineering using annulus fibrosus (AF) and nucleus pulposus (NP) cells in relation to potential clinical application. PLGA scaffolds were soaked in cells-fibrin suspension and polymerized by dropping thrombin-sodium chloride (CaCl(2)) solution. A PLGA-cell complex without fibrin was used as control. Higher cellular proliferation activity was observed in fibrin/PLGA-seeded AF and NP cells at each time point of 3, 7, 14 and 7 days using the MTT assay. After 3 weeks in vitro incubation, fibrin/PLGA exhibited a firmer gross morphology than PLGA groups. A significant cartilaginous tissue formation was observed in fibrin/PLGA, as proven by the development of cells cluster of various sizes and three-dimensional (3D) cartilaginous histoarchitecture and the presence of proteoglycan-rich matrix and glycosaminoglycan (GAG). The sGAG production measured by 1,9-dimethylmethylene blue (DMMB) assay revealed greater sGAG production in fibrin/PLGA than PLGA group. Immunohistochemical analyses showed expressions of collagen type II, aggrecan core protein and collagen type I genes throughout in vitro culture in both fibrin/PLGA and PLGA. In conclusion, fibrin promotes cell proliferation, stable in vitro tissue morphology, superior cartilaginous tissue formation and sGAG production of AF and NP cells cultured in PLGA scaffold. The 3D porous PLGA scaffold-cell complexes using fibrin can provide a vehicle for delivery of cells to regenerate tissue-engineered IVD tissue.

Matched MeSH terms: Collagen Type II/genetics
The use of fibrin and poly(lactic-co-glycolic acid) hybrid scaffold for articular cartilage tissue engineering: an in vivo analysis

Munirah S, Kim SH, Ruszymah BH, Khang G

Eur Cell Mater, 2008 Feb 21;15:41-52.
PMID: 18288632

Our preliminary results indicated that fibrin and poly(lactic-co-glycolic acid) (PLGA) hybrid scaffold promoted early chondrogenesis of articular cartilage constructs in vitro. The aim of this study was to evaluate in vivo cartilaginous tissue formation by chondrocyte-seeded fibrin/PLGA hybrid scaffolds. PLGA scaffolds were soaked carefully, in chondrocyte-fibrin suspension, and polymerized by dropping thrombin-calcium chloride (CaCl2) solution. PLGA-seeded chondrocytes were used as a control. Resulting constructs were implanted subcutaneously, at the dorsum of nude mice, for 4 weeks. Macroscopic observation, histological evaluation, gene expression and sulphated-glycosaminoglycan (sGAG) analyses were performed at each time point of 1, 2 and 4 weeks post-implantation. Cartilaginous tissue formation in fibrin/PLGA hybrid construct was confirmed by the presence of lacunae and cartilage-isolated cells embedded within basophilic ground substance. Presence of proteoglycan and glycosaminoglycan (GAG) in fibrin/PLGA hybrid constructs was confirmed by positive Safranin O and Alcian Blue staining. Collagen type II exhibited intense immunopositivity at the pericellular matrices. Chondrogenic properties were further demonstrated by the expression of gene encoded cartilage-specific markers, collagen type II and aggrecan core protein. The sGAG production in fibrin/PLGA hybrid constructs was higher than in the PLGA group. In conclusion, fibrin/PLGA hybrid scaffold promotes cartilaginous tissue formation in vivo and may serve as a potential cell delivery vehicle and a structural basis for articular cartilage tissue-engineering.

Matched MeSH terms: Collagen Type II/genetics
Gene expression characteristic in human auricular cartilage tissue engineering

Farah Wahida I, Aminuddin BS, Munirah S, Chua KH, Fuzina NH, Isa MR, et al.

Med J Malaysia, 2004 May;59 Suppl B:190-1.
PMID: 15468882

This study was to assess collagen type II and collagen type I gene expression in tissue-engineered human auricular: cartilage formed via tissue engineering technique. Large-scale culture expansions were transformed into 3D in vitro construct and were implanted subcutaneously on the dorsal of athymic mice. After 8 weeks, explanted construct was processed in the same manner of native cartilage to facilitate cells for gene expression analysis. Isolated cells from in vivo construct demonstrated expression of type II collagen gene comparable to native cartilage. This study verified that tissue-engineered auricular cartilage expressed cartilage specific gene, collagen type II after in vivo maturation.

Matched MeSH terms: Collagen Type II/genetics*
Human serum provided additional values in growth factors supplemented medium for human chondrocytes monolayer expansion and engineered cartilage construction

Chua KH, Aminuddin BS, Fuzina NH, Ruszymah BH

Med J Malaysia, 2004 May;59 Suppl B:194-5.
PMID: 15468884

We have previously formulated an optimized human chondrocytes growth medium based on 2% fetal bovine serum supplementation. For clinical usage, the animal serum must be replaced by patient own serum. We investigated the effects of human serum concentration for human nasal septum chondrocytes monolayer culture and cartilage reconstruction. Human serum demonstrated a dose dependent manner in promoting chondrocytes growth and cartilage engineering.

Matched MeSH terms: Collagen Type II/genetics
Insulin-transferrin-selenium prevent human chondrocyte dedifferentiation and promote the formation of high quality tissue engineered human hyaline cartilage

Chua KH, Aminuddin BS, Fuzina NH, Ruszymah BH

Eur Cell Mater, 2005 Jun 17;9:58-67; discussion 67.
PMID: 15962238

This study was to investigate the effects of insulin-transferrin-selenium (ITS) on the proliferation and quantitative gene expression of adult human nasal septum chondrocytes in monolayer culture expansion and the formation of tissue engineered hyaline cartilage. Effects of ITS on human nasal septum chondrocytes monolayer culture expansion and gene expression were evaluated in various culture media either added with 2% fetal bovine serum (FBS) or 1 ng/mL basic fibroblast growth factor plus 1 ng/mL transforming growth factor or both serum and growth factors supplementation in comparison with medium added with 10%FBS. Chondrocytes cultured in medium added with 2% fetal bovine serum and growth factors either supplemented with or without ITS were then mixed with pluronic F-127 hydrogel for in vivo tissue engineered cartilage formation in nude mice model. Engineered tissues were removed after 8 weeks of implantation and evaluated with histological staining, immunohistochemistry, transmission electron microscopy and quantitative gene expression analysis. ITS promoted human chondrocytes proliferation and reduced chondrocytes dedifferentiation in media supplemented with serum and growth factors. ITS with 2% FBS and growth factors provided 15-fold increased in chondrocytes number by the end of the culture period compared to the standard culture medium used in chondrocytes culture (medium added with 10% FBS). Engineered tissue resulted from ITS supplementation demonstrated higher quality of cartilage formation. In conclusion, our study has demonstrated the benefits of ITS supplementation in human chondrocytes monolayer culture and tissue engineering cartilage formation.

Matched MeSH terms: Collagen Type II/genetics