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Ex vivo expanded SSEA-4+ human limbal stromal cells are multipotent and do not express other embryonic stem cell markers

Lim MN, Hussin NH, Othman A, Umapathy T, Baharuddin P, Jamal R, et al.

Mol Vis, 2012;18:1289-300.
PMID: 22665977

The presence of multipotent human limbal stromal cells resembling mesenchymal stromal cells (MSC) provides new insights to the characteristic of these cells and its therapeutic potential. However, little is known about the expression of stage-specific embryonic antigen 4 (SSEA-4) and the embryonic stem cell (ESC)-like properties of these cells. We studied the expression of SSEA-4 surface protein and the various ESC and MSC markers in the ex vivo cultured limbal stromal cells. The phenotypes and multipotent differentiation potential of these cells were also evaluated.

Matched MeSH terms: Chondrocytes/cytology
Fulltext Development of a cartilage composite utilizing porous tantalum, fibrin, and rabbit chondrocytes for treatment of cartilage defect

Jamil K, Chua KH, Joudi S, Ng SL, Yahaya NH

J Orthop Surg Res, 2015;10:27.
PMID: 25889942 DOI: 10.1186/s13018-015-0166-z

Functional tissue engineering has emerged as a potential means for treatment of cartilage defect. Development of a stable cartilage composite is considered to be a good option. The aim of the study was to observe whether the incorporation of cultured chondrocytes on porous tantalum utilizing fibrin as a cell carrier would promote cartilage tissue formation.

Matched MeSH terms: Chondrocytes/cytology
Generating neuron-like cells from BM-derived mesenchymal stromal cells in vitro

Choong PF, Mok PL, Cheong SK, Leong CF, Then KY

Cytotherapy, 2007;9(2):170-83.
PMID: 17453969

The multipotency of stromal cells has been studied extensively. It has been reported that mesenchymal stromal cells (MSC) are capable of differentiating into cells of multilineage. Different methods and reagents have been used to induce the differentiation of MSC. We investigated the efficacy of different growth factors in inducing MSC differentiation into neurons.

Matched MeSH terms: Chondrocytes/cytology
Fulltext Genipin crosslinked chitosan/PEO nanofibrous scaffolds exhibiting an improved microenvironment for the regeneration of articular cartilage

Ching KY, Andriotis O, Sengers B, Stolz M

J Biomater Appl, 2021 09;36(3):503-516.
PMID: 33730922 DOI: 10.1177/08853282211002015

Towards optimizing the growth of extracellular matrix to produce repair cartilage for healing articular cartilage (AC) defects in joints, scaffold-based tissue engineering approaches have recently become a focus of clinical research. Scaffold-based approaches by electrospinning aim to support the differentiation of chondrocytes by providing an ultrastructure similar to the fibrillar meshwork in native cartilage. In a first step, we demonstrate how the blending of chitosan with poly(ethylene oxide) (PEO) allows concentrated chitosan solution to become electrospinnable. The chitosan-based scaffolds share the chemical structure and characteristics of glycosaminoglycans, which are important structural components of the cartilage extracellular matrix. Electrospinning produced nanofibrils of ∼100 nm thickness that are closely mimicking the size of collagen fibrils in human AC. The polymer scaffolds were stabilized in physiological conditions and their stiffness was tuned by introducing the biocompatible natural crosslinker genipin. We produced scaffolds that were crosslinked with 1.0% genipin to obtain values of stiffness that were in between the stiffness of the superficial zone human AC of 600 ± 150 kPa and deep zone AC of 1854 ± 483 kPa, whereas the stiffness of 1.5% genipin crosslinked scaffold was similar to the stiffness of deep zone AC. The scaffolds were degradable, which was indicated by changes in the fibril structure and a decrease in the scaffold stiffness after seven months. Histological and immunohistochemical analysis after three weeks of culture with human articular chondrocytes (HACs) showed a cell viability of over 90% on the scaffolds and new extracellular matrix deposited on the scaffolds.

Matched MeSH terms: Chondrocytes/cytology
Towards engineering of a tracheal equivalent: identification of epithelial precursor cells, differentiation and cocultivation techniques

Rotter N, Stölzel K, Endres M, Leinhase I, Ziegelaar BW, Sittinger M

Med J Malaysia, 2004 May;59 Suppl B:35-6.
PMID: 15468806
Matched MeSH terms: Chondrocytes/cytology
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: Chondrocytes/cytology
The fundamentals of tissue engineering: new scaffolds

Di Silvio L, Gurav N, Sambrook R

Med J Malaysia, 2004 May;59 Suppl B:89-90.
PMID: 15468832

The ability to regenerate new bone for skeletal use is a major clinical need. In this study, two novel porous calcium phosphate materials pure HA and biphasic HA/beta-Tricalcium phosphate (HA/beta -TCP) were evaluated as potential scaffolds for cell-seeded bone substitutes using human osteoblast-like cells (HOS) and primary human mesenchymal stem cells (hMSCs). A high rate of proliferation was observed on both scaffolds. A greater increase in alkaline phosphatase (ALP- an indicator of osteoblast differentiation) was observed on HA/beta -TCP compared to HA. This observation indicates that HA/TCP may play a role in inducing osteoblastic differentiation. Although further evaluation is required both materials show potential as innovative synthetic substitutes for tissue engineered scaffolds.

Matched MeSH terms: Chondrocytes/cytology
Expansion of human articular chondrocytes and formation of tissue-engineered cartilage: a step towards exploring a potential use of matrix-induced cell therapy

Munirah S, Samsudin OC, Aminuddin BS, Ruszymah BH

Tissue Cell, 2010 Oct;42(5):282-92.
PMID: 20810142 DOI: 10.1016/j.tice.2010.07.002

Monolayer culture expansion remains as a fundamental step to acquire sufficient number of cells for 3D constructs formation. It has been well-documented that cell expansion is however accompanied by cellular dedifferentiation. In order to promote cell growth and circumvent cellular dedifferentiation, we evaluated the effects of Transforming Growth Factor Beta-2 (TGF-β2), Insulin-like Growth Factor-I (IGF-I) and basic Fibroblast Growth Factor (bFGF) combination on articular chondrocytes culture and 'chondrocytes-fibrin' construct formation. Chondrocytes were serially cultured in: (1) F12:DMEM+10% Foetal Bovine Serum (FBS) with growth factors (FD10GFs), (2) F12:DMEM+2%FBS with the growth factors (FD2GFs) and, (3) F12:DMEM+10%FBS without growth factors (FD) as control. Cultured chondrocytes were evaluated by means of growth kinetics parameters, cell cycle analysis, quantitative phenotypic expression of collagen type II, aggrecan core protein sox-9 and collagen type I and, immunochemistry technique. Harvested chondrocytes were incorporated with plasma-derived fibrin and were polymerized to form the 3D constructs and implanted subcutaneously at the dorsum of athymic nude mice for eight (8) weeks. Resulted constructs were assigned for gross inspections and microscopic evaluation using standard histochemicals staining, immunochemistry technique and, quantitative phenotypic expression of cartilage markers to reassure cartilaginous tissue formation. Growth kinetics performance of chondrocytes cultured in three (3) types of culture media from the most to least was in the following order: FD10GFs>FD2GFs>FD. Following growth kinetics analysis, we decided to use FD10GFs and FD (control) for further evaluation and 'chondrocytes-fibrin' constructs formation. Chondrocytes cultured in FD10GFs preserved the normal diploid state (2c) with no evidence of aneuploidy, haploidy or tetraploidy. Expression of cartilage-specific markers namely collagen type II, aggrecan core protein and sox-9 were significantly higher in FD10GFs when compared to control. After implantation, 'chondrocytes-fibrin' constructs exhibited firm, white, smooth and glistening cartilage-like properties. FD10GFs constructs formed better quality cartilage-like tissue than FD constructs in term of overall cartilaginous tissue formation, cells organization and extracellular matrix distribution in the specimens. Cartilaginous tissue formation was confirmed by the presence of lacunae and cartilage-isolated cells embedded within basophilic ground substance. Presence of proteoglycan was confirmed by positive Safranin O staining. Collagen type II exhibited immunopositivity at the pericellular and inter-territorial matrix area. Chondrogenic properties of the construct were further confirmed by the expression of genes encoding collagen type II, aggrecan core protein and sox9. In conclusion, FD10GFs promotes the proliferation of chondrocytes and formation of good quality 'chondrocytes-fibrin' constructs which may have potential use of matrix-induced cell implantation.

Matched MeSH terms: Chondrocytes/cytology*
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: Chondrocytes/cytology*
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: Chondrocytes/cytology*
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: Chondrocytes/cytology*
Formation of tissue-engineered human auricular cartilage via tissue engineering technique for future use in ear surgery

Saim L, Aminuddin BS, Munirah S, Chua KH, Izuddin Fahmy A, Fuzina NH, et al.

Med J Malaysia, 2004 May;59 Suppl B:192-3.
PMID: 15468883

To date there is no optimal approach to reconstruct an external ear. However, advances in tissue engineering technologies have indicated that in vitro autologous elastic cartilage might be of great importance in the future treatment of these patients. The aim of this study was to observe monolayer expansion of auricular cartilage and to evaluate engineered cartilage using standard histochemical study.

Matched MeSH terms: Chondrocytes/cytology*
In vitro development of autologous tissue engineered human articular neocartilage for orthopaedic surgery

Samsudin OC, Aminuddin BS, Munirah S, Chua KH, Fuzina NH, Isa MR, et al.

Med J Malaysia, 2004 May;59 Suppl B:15-6.
PMID: 15468796

Treatment of articular cartilage lesions remains a clinical challenge. The uses of prosthetic joint replace allograft and/or autograft transplant carry a risk of complications due to infection, loosening of its component, immunological rejection and morbidity at the donor site. There has been an increasing interest in the management of cartilage damages, owing to the introduction of new therapeutic options. Tissue engineering as a method for tissue restoration begins to provide a potential alternative therapy for autologous grafts transplantations. We aimed to evaluate how well a tissue engineered neocartilage implant, consist of human articular chondrocytes cultured with the presence of autologous serum and mixed in a fresh fibrin derived from patient, would perform in subcutaneous implantation in athymic mice.

Matched MeSH terms: Chondrocytes/cytology*
The significance of using pooled human serum in human articular cartilage tissue engineering

Azmi B, Aminuddin BS, Sharaf I, Samsudin OC, Munirah S, Chua KH, et al.

Med J Malaysia, 2004 May;59 Suppl B:13-4.
PMID: 15468795

Animal serum is commonly used in chondrocytes culture expansion to promote cell proliferation and shorten the time lag before new tissue reconstruction is possible. However, animal serum is not suitable for regeneration of clinical tissue because it has potential risk of viral and prion related disease transmission particularly mad cow disease and foreign protein contamination that can stimulate immune reaction leading to graft rejection. In this context, human serum as homologous supplement has a greater potential as growth promoting agents for human chondrocytes culture.

Matched MeSH terms: Chondrocytes/cytology*
Tissue engineered human articular neocartilage using serial expanded chondrocytes

Munirah S, Aminuddin BS, Chua KH, Fuzina NH, Isa MR, Ruszymah BH

Med J Malaysia, 2004 May;59 Suppl B:9-10.
PMID: 15468793

Autologous cells are usually preferred in treating damaged tissue to avoid risks of immunological rejection and transmitting infectious diseases. Since only limited amount of tissue can be obtained without causing morbidity at the donor site, in vitro expansion of isolated cell is essential in order to acquire sufficient number of cells to reconstruct neocartilage. The aim of this study was to examine whether serial expanded chondrocytes can be use to generate neocartilage in vivo.

Matched MeSH terms: Chondrocytes/cytology*
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: Chondrocytes/cytology*
The effects of autologous human serum on the growth of tissue engineered human articular cartilage

Badrul AH, Aminuddin BS, Sharaf I, Samsudin OC, Munirah S, Ruszymah BH

Med J Malaysia, 2004 May;59 Suppl B:11-2.
PMID: 15468794

Culture media supplemented with animal serum e.g. fetal bovine serum; FBS is commonly used for human culture expansion. However, for clinical application, FBS is restricted as its carry a risk of viral or prion transmission. Engineering autologous cartilage with autologous human serum supplementation is seen as a better solution to reduce the risk of transmitting infectious diseases and immune rejection during cartilage transplantation. The purpose of this study is to establish and compare the effects of 10% autologous human serum (AHS) and 10% FBS on the growth of chondrocytes and the formation of tissue engineered human articular cartilage.

Matched MeSH terms: Chondrocytes/cytology*
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: Chondrocytes/cytology*
Fulltext Basic fibroblast growth factor with human serum supplementation: enhancement of human chondrocyte proliferation and promotion of cartilage regeneration

Chua KH, Aminuddin BS, Fuzina NH, Ruszymah BH

Singapore Med J, 2007 Apr;48(4):324-32.
PMID: 17384880

The objectives of this study were to determine the optimum concentration of basic fibroblast growth factor (bFGF) in foetal bovine serum (FBS) or human serum (HS) supplemented medium for adult human nasal septum chondrocyte culture and to evaluate the potential of cartilage regeneration.

Matched MeSH terms: Chondrocytes/cytology
Isolation, Expansion, and Characterization of Wharton's Jelly-Derived Mesenchymal Stromal Cell: Method to Identify Functional Passages for Experiments

Aung SW, Abu Kasim NH, Ramasamy TS

Methods Mol Biol, 2019;2045:323-335.
PMID: 31201682 DOI: 10.1007/7651_2019_242

The therapeutic potential of human mesenchymal stromal stem cells (hMSCs) for cell-based therapeutic is greatly influenced by the in vitro culture condition including the culture conditions. Nevertheless, there are many technical challenges needed to be overcome prior to the clinical use including the quantity, quality, and heterogeneity of the cells. Therefore, it is necessary to develop a stem cell culture procedure or protocol for cell expansion in order to generate reproducible and high-quality cells in accordance with good manufacturing practice for clinical and therapeutic purposes. Here we assessed the MSCs characteristic of human Wharton's jelly mesenchymal stromal cells in in vitro culture according to the criteria established by the International Society for Cellular Therapy. Besides, the viability of the WJMSCs was determined in order to increase the confidence that the cells are employed to meet the therapeutic efficacy.

Matched MeSH terms: Chondrocytes/cytology

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