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  1. 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: Cartilage, Articular/cytology
  2. 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: Cartilage, Articular/cytology
  3. Ab-Rahim S, Selvaratnam L, Kamarul T
    Cell Biol Int, 2008 Jul;32(7):841-7.
    PMID: 18479947 DOI: 10.1016/j.cellbi.2008.03.016
    Articular cartilage extracellular matrix (ECM) plays a crucial role in regulating chondrocyte functions via cell-matrix interaction, cytoskeletal organization and integrin-mediated signaling. Factors such as interleukins, basic fibroblast growth factor (bFGF), bone morphogenic proteins (BMPs) and insulin-like growth factor (IGF) have been shown to modulate the synthesis of extracellular matrix in vitro. However, the effects of TGF-beta1 and beta-estradiol in ECM regulation require further investigation, although there have been suggestions that these factors do play a positive role. To establish the role of these factors on chondrocytes derived from articular joints, a study was conducted to investigate the effects of TGF-beta1 and beta-estradiol on glycosaminoglycan secretion and type II collagen distribution (two major component of cartilage ECM in vivo). Thus, chondrocyte cultures initiated from rabbit articular cartilage were treated with 10ng/ml of TGF-beta1, 10nM of beta-estradiol or with a combination of both factors. Sulphated glycosaminoglycan (GAG) and type II collagen levels were then measured in both these culture systems. The results revealed that the synthesis of GAG and type II collagen was shown to be enhanced in the TGF-beta1 treated cultures. This increase was also noted when TGF-beta1 and beta-estradiol were both used as culture supplements. However, beta-estradiol alone did not appear to affect GAG or type II collagen deposition. There was also no difference between the amount of collagen type II and GAG being expressed when chondrocyte cultures were treated with TGF-beta1 when compared with cultures treated with combined factors. From this, we conclude that although TGF-beta1 appears to stimulate chondrocyte ECM synthesis, beta-estradiol fails to produce similar effects. The findings of this study confirm that contrary to previous claims, beta-estradiol has little or no effect on chondrocyte ECM synthesis. Furthermore, the use of TGF-beta1 may be useful in future studies looking into biological mechanisms by which ECM synthesis in chondrocyte cultures can be augmented, particularly for clinical application.
    Matched MeSH terms: Cartilage, Articular/cytology
  4. Munirah S, Ruszymah BH, Samsudin OC, Badrul AH, Azmi B, Aminuddin BS
    J Orthop Surg (Hong Kong), 2008 Aug;16(2):220-9.
    PMID: 18725677
    To evaluate the effect of autologous human serum (AHS) versus pooled human serum (PHS) versus foetal bovine serum (FBS) for growth of articular chondrocytes and formation of chondrocytefibrin constructs.
    Matched MeSH terms: Cartilage, Articular/cytology
  5. Kamarul T, Selvaratnam L, Masjuddin T, Ab-Rahim S, Ng C, Chan KY, et al.
    J Orthop Surg (Hong Kong), 2008 Aug;16(2):230-6.
    PMID: 18725678
    To compare the efficacy of autologous chondrocyte transplantation (ACT) versus non-operative measures for cartilage repair in rabbits.
    Matched MeSH terms: Cartilage, Articular/cytology
  6. Saw KY, Hussin P, Loke SC, Azam M, Chen HC, Tay YG, et al.
    Arthroscopy, 2009 Dec;25(12):1391-400.
    PMID: 19962065 DOI: 10.1016/j.arthro.2009.07.011
    PURPOSE: The purpose of the study was to determine whether postoperative intra-articular injections of autologous marrow aspirate (MA) and hyaluronic acid (HA) after subchondral drilling resulted in better cartilage repair as assessed histologically by Gill scoring.
    METHODS: In a goat model we created a 4-mm full-thickness articular cartilage defect in the stifle joint (equivalent to 1.6 cm in the human knee) and conducted subchondral drilling. The animals were divided into 3 groups: group A (control), no injections; group B (HA), weekly injection of 1 mL of sodium hyaluronate for 3 weeks; and group C (HA + MA), similar to group B but with 2 mL of autologous MA in addition to HA. MA was obtained by bone marrow aspiration, centrifuged, and divided into aliquots for cryopreservation. Fifteen animals were equally divided between the groups and sacrificed 24 weeks after surgery, when the joint was harvested, examined macroscopically and histologically.
    RESULTS: Of the 15 animals, 2 from group A had died of non-surgery-related complications and 1 from group C was excluded because of a joint infection. In group A the repair constituted mainly scar tissue, whereas in group B there was less scar tissue, with small amounts of proteoglycan and type II collagen at the osteochondral junction. In contrast, repair cartilage from group C animals showed almost complete coverage of the defect with evidence of hyaline cartilage regeneration. Histology assessed by Gill scoring was significantly better in group C with 1-way analysis of variance yielding an F statistic of 10.611 with a P value of .004, which was highly significant.
    CONCLUSIONS: Postoperative intra-articular injections of autologous MA in combination with HA after subchondral drilling resulted in better cartilage repair as assessed histologically by Gill scoring in a goat model.
    CLINICAL RELEVANCE: After arthroscopic subchondral drilling, this novel technique may result in better articular cartilage regeneration.
    Matched MeSH terms: Cartilage, Articular/cytology
  7. 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: Cartilage, Articular/cytology*
  8. Moo EK, Osman NA, Pingguan-Murphy B
    Clinics (Sao Paulo), 2011;66(8):1431-6.
    PMID: 21915496
    INTRODUCTION: Although previous studies have been performed on cartilage explant cultures, the generalized dynamics of cartilage metabolism after extraction from the host are still poorly understood due to differences in the experimental setups across studies, which in turn prevent building a complete picture.

    METHODS: In this study, we investigated the response of cartilage to the trauma sustained during extraction and determined the time needed for the cartilage to stabilize. Explants were extracted aseptically from bovine metacarpal-phalangeal joints and cultured for up to 17 days.

    RESULTS: The cell viability, cell number, proteoglycan content, and collagen content of the harvested explants were analyzed at 0, 2, 10, and 17 days after explantation. A high percentage of the cartilage explants were found to be viable. The cell density initially increased significantly but stabilized after two days. The proteoglycan content decreased gradually over time, but it did not decrease to a significant level due to leakage through the distorted peripheral collagen network and into the bathing medium. The collagen content remained stable for most of the culture period until it dropped abruptly on day 17.

    CONCLUSION: Overall, the tested cartilage explants were sustainable over long-term culture. They were most stable from day 2 to day 10. The degradation of the collagen on day 17 did not reach diseased levels, but it indicated the potential of the cultures to develop into degenerated cartilage. These findings have implications for the application of cartilage explants in pathophysiological fields.

    Matched MeSH terms: Cartilage, Articular/cytology
  9. Hamid AA, Idrus RB, Saim AB, Sathappan S, Chua KH
    Clinics (Sao Paulo), 2012;67(2):99-106.
    PMID: 22358233
    OBJECTIVES: Understanding the changes in chondrogenic gene expression that are involved in the differentiation of human adipose-derived stem cells to chondrogenic cells is important prior to using this approach for cartilage repair. The aims of the study were to characterize human adipose-derived stem cells and to examine chondrogenic gene expression after one, two, and three weeks of induction.

    MATERIALS AND METHODS: Human adipose-derived stem cells at passage 4 were evaluated by flow cytometry to examine the expression of surface markers. These adipose-derived stem cells were tested for adipogenic and osteogenic differentiation capacity. Ribonucleic acid was extracted from the cells for quantitative polymerase chain reaction analysis to determine the expression levels of chondrogenic genes after chondrogenic induction.

    RESULTS: Human adipose-derived stem cells were strongly positive for the mesenchymal markers CD90, CD73, CD44, CD9, and histocompatibility antigen and successfully differentiated into adipogenic and osteogenic lineages. The human adipose-derived stem cells aggregated and formed a dense matrix after chondrogenic induction. The expression of chondrogenic genes (collagen type II, aggrecan core protein, collagen type XI, COMP, and ELASTIN) was significantly higher after the first week of induction. However, a significantly elevated expression of collagen type X was observed after three weeks of chondrogenic induction.

    CONCLUSION: Human adipose-derived stem cells retain stem cell characteristics after expansion in culture to passage 4 and serve as a feasible source of cells for cartilage regeneration. Chondrogenesis in human adipose-derived stem cells was most prominent after one week of chondrogenic induction.

    Matched MeSH terms: Cartilage, Articular/cytology*
  10. Ab-Rahim S, Selvaratnam L, Raghavendran HR, Kamarul T
    Mol Cell Biochem, 2013 Apr;376(1-2):11-20.
    PMID: 23238871 DOI: 10.1007/s11010-012-1543-0
    Tissue engineering approaches often require expansion of cell numbers in vitro to accelerate tissue regenerative processes. Although several studies have used this technique for therapeutic purposes, a major concern involving the use of isolated chondrocyte culture is the reduction of extracellular matrix (ECM) protein expressed due to the transfer of cells from the normal physiological milieu to the artificial 2D environment provided by the cell culture flasks. To overcome this issue, the use of alginate hydrogel beads as a substrate in chondrocyte cultures has been suggested. However, the resultant characteristics of cells embedded in this bead is elusive. To elucidate this, a study using chondrocytes isolated from rabbit knee articular cartilage expanded in vitro as monolayer and chondrocyte-alginate constructs was conducted. Immunohistochemical evaluation and ECM distribution was examined with or without transforming growth factor (TGF-β1) supplement to determine the ability of cells to express major chondrogenic proteins in these environments. Histological examination followed by transmission electron microscopy and scanning electron microscopy was performed to determine the morphology and the ultrastructural characteristics of these cells. Results demonstrated a significant increase in glycosaminoglycan/mg protein levels in chondrocyte cultures grown in alginate construct than in monolayer cultures. In addition, an abundance of ECM protein distribution surrounding chondrocytes cultured in alginate hydrogel was observed. In conclusion, the current study demonstrates that the use of alginate hydrogel beads in chondrocyte cultures with or without TGF-β1 supplement provided superior ECM expression than monolayer cultures.
    Matched MeSH terms: Cartilage, Articular/cytology
  11. Moo EK, Abusara Z, Abu Osman NA, Pingguan-Murphy B, Herzog W
    J Biomech, 2013 Aug 9;46(12):2024-31.
    PMID: 23849134 DOI: 10.1016/j.jbiomech.2013.06.007
    Morphological studies of live connective tissue cells are imperative to helping understand cellular responses to mechanical stimuli. However, photobleaching is a constant problem to accurate and reliable live cell fluorescent imaging, and various image thresholding methods have been adopted to account for photobleaching effects. Previous studies showed that dual photon excitation (DPE) techniques are superior over conventional one photon excitation (OPE) confocal techniques in minimizing photobleaching. In this study, we investigated the effects of photobleaching resulting from OPE and DPE on morphology of in situ articular cartilage chondrocytes across repeat laser exposures. Additionally, we compared the effectiveness of three commonly-used image thresholding methods in accounting for photobleaching effects, with and without tissue loading through compression. In general, photobleaching leads to an apparent volume reduction for subsequent image scans. Performing seven consecutive scans of chondrocytes in unloaded cartilage, we found that the apparent cell volume loss caused by DPE microscopy is much smaller than that observed using OPE microscopy. Applying scan-specific image thresholds did not prevent the photobleaching-induced volume loss, and volume reductions were non-uniform over the seven repeat scans. During cartilage loading through compression, cell fluorescence increased and, depending on the thresholding method used, led to different volume changes. Therefore, different conclusions on cell volume changes may be drawn during tissue compression, depending on the image thresholding methods used. In conclusion, our findings confirm that photobleaching directly affects cell morphology measurements, and that DPE causes less photobleaching artifacts than OPE for uncompressed cells. When cells are compressed during tissue loading, a complicated interplay between photobleaching effects and compression-induced fluorescence increase may lead to interpretations in cell responses to mechanical stimuli that depend on the microscopic approach and the thresholding methods used and may result in contradictory interpretations.
    Matched MeSH terms: Cartilage, Articular/cytology
  12. Chua KH, Lee TH, Nagandran K, Md Yahaya NH, Lee CT, Tjih ET, et al.
    PMID: 23339380 DOI: 10.1186/1472-6882-13-19
    Osteoarthritis (OA) is a degenerative joint disease that results in the destruction of cartilage. Edible Bird's Nest (EBN) extract contains important components, which can reduce the progression of osteoarthritis and helps in the regeneration of the cartilage. The present study aimed to investigate the effect of EBN extract on the catabolic and anabolic activities of the human articular chondrocytes (HACs) isolated from the knee joint of patients with OA.
    Matched MeSH terms: Cartilage, Articular/cytology
  13. Choi JR, Yong KW, Choi JY
    J Cell Physiol, 2018 Mar;233(3):1913-1928.
    PMID: 28542924 DOI: 10.1002/jcp.26018
    Today, articular cartilage damage is a major health problem, affecting people of all ages. The existing conventional articular cartilage repair techniques, such as autologous chondrocyte implantation (ACI), microfracture, and mosaicplasty, have many shortcomings which negatively affect their clinical outcomes. Therefore, it is essential to develop an alternative and efficient articular repair technique that can address those shortcomings. Cartilage tissue engineering, which aims to create a tissue-engineered cartilage derived from human mesenchymal stem cells (MSCs), shows great promise for improving articular cartilage defect therapy. However, the use of tissue-engineered cartilage for the clinical therapy of articular cartilage defect still remains challenging. Despite the importance of mechanical loading to create a functional cartilage has been well demonstrated, the specific type of mechanical loading and its optimal loading regime is still under investigation. This review summarizes the most recent advances in the effects of mechanical loading on human MSCs. First, the existing conventional articular repair techniques and their shortcomings are highlighted. The important parameters for the evaluation of the tissue-engineered cartilage, including chondrogenic and hypertrophic differentiation of human MSCs are briefly discussed. The influence of mechanical loading on human MSCs is subsequently reviewed and the possible mechanotransduction signaling is highlighted. The development of non-hypertrophic chondrogenesis in response to the changing mechanical microenvironment will aid in the establishment of a tissue-engineered cartilage for efficient articular cartilage repair.
    Matched MeSH terms: Cartilage, Articular/cytology
  14. Muhammad SA, Nordin N, Mehat MZ, Fakurazi S
    Cell Tissue Res, 2019 Feb;375(2):329-344.
    PMID: 30084022 DOI: 10.1007/s00441-018-2884-0
    Articular cartilage defect remains the most challenging joint disease due to limited intrinsic healing capacity of the cartilage that most often progresses to osteoarthritis. In recent years, stem cell therapy has evolved as therapeutic strategies for articular cartilage regeneration. However, a number of studies have shown that therapeutic efficacy of stem cell transplantation is attributed to multiple secreted factors that modulate the surrounding milieu to evoke reparative processes. This systematic review and meta-analysis aim to evaluate and compare the therapeutic efficacy of stem cell and secretome in articular cartilage regeneration in animal models. We systematically searched the PubMed, CINAHL, Cochrane Library, Ovid Medline and Scopus databases until August 2017 using search terms related to stem cells, cartilage regeneration and animals. A random effect meta-analysis of the included studies was performed to assess the treatment effects on new cartilage formation on an absolute score of 0-100% scale. Subgroup analyses were also performed by sorting studies independently based on similar characteristics. The pooled analysis of 59 studies that utilized stem cells significantly improved new cartilage formation by 25.99% as compared with control. Similarly, the secretome also significantly increased cartilage regeneration by 26.08% in comparison to the control. Subgroup analyses revealed no significant difference in the effect of stem cells in new cartilage formation. However, there was a significant decline in the effect of stem cells in articular cartilage regeneration during long-term follow-up, suggesting that the duration of follow-up is a predictor of new cartilage formation. Secretome has shown a similar effect to stem cells in new cartilage formation. The risk of bias assessment showed poor reporting for most studies thereby limiting the actual risk of bias assessment. The present study suggests that both stem cells and secretome interventions improve cartilage regeneration in animal trials. Graphical abstract ᅟ.
    Matched MeSH terms: Cartilage, Articular/cytology*
  15. 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: Cartilage, Articular/cytology
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