Displaying publications 61 - 80 of 101 in total

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  1. Fulltext A preliminary study comparing the use of allogenic chondrogenic pre-differentiated and undifferentiated mesenchymal stem cells for the repair of full thickness articular cartilage defects in rabbits
    Dashtdar H, Rothan HA, Tay T, Ahmad RE, Ali R, Tay LX, et al.
    J Orthop Res, 2011 Sep;29(9):1336-42.
    PMID: 21445989 DOI: 10.1002/jor.21413
    Chondrogenic differentiated mesenchymal stem cells (CMSCs) have been shown to produce superior chondrogenic expression markers in vitro. However, the use of these cells in vivo has not been fully explored. In this study, in vivo assessment of cartilage repair potential between allogenic-derived chondrogenic pre-differentiated mesenchymal stem cells and undifferentiated MSCs (MSCs) were compared. Bilateral full thickness cartilage defects were created on the medial femoral condyles of 12 rabbits (n = 12). Rabbits were divided into two groups. In one group, the defects in the right knees were repaired using alginate encapsulated MSCs while in the second group, CMSCs were used. The animals were sacrificed and the repaired and control knees were assessed at 3 and 6 months after implantation. Quantitative analysis was performed by measuring the Glycosaminoglycans (GAGs)/total protein content. The mean Brittberg score was higher in the transplanted knees as compared to the untreated knee at 6 months (p  0.05). This study demonstrates that the use of either MSC or CMSC produced superior healing when compared to cartilage defects that were untreated. However, both cells produced comparable treatment outcomes.
    Matched MeSH terms: Chondrocytes/cytology*; Chondrocytes/transplantation
  2. Fulltext Human peripheral blood derived mesenchymal stem cells demonstrate similar characteristics and chondrogenic differentiation potential to bone marrow derived mesenchymal stem cells
    Chong PP, Selvaratnam L, Abbas AA, Kamarul T
    J Orthop Res, 2012 Apr;30(4):634-42.
    PMID: 21922534 DOI: 10.1002/jor.21556
    The use of mesenchymal stem cells (MSCs) for cartilage repair has generated much interest owing to their multipotentiality. However, their significant presence in peripheral blood (PB) has been a matter of much debate. The objectives of this study are to isolate and characterize MSCs derived from PB and, compare their chondrogenic potential to MSC derived from bone marrow (BM). PB and BM derived MSCs from 20 patients were isolated and characterized. From 2 ml of PB and BM, 5.4 ± 0.6 million and 10.5 ± 0.8 million adherent cells, respectively, were obtained by cell cultures at passage 2. Both PB and BM derived MSCs were able to undergo tri-lineage differentiation and showed negative expression of CD34 and CD45, but positively expressed CD105, CD166, and CD29. Qualitative and quantitative examinations on the chondrogenic potential of PB and BM derived MSCs expressed similar cartilage specific gene (COMP) and proteoglycan levels, respectively. Furthermore, the s-GAG levels expressed by chondrogenic MSCs in cultures were similar to that of native chondrocytes. In conclusion, this study demonstrates that MSCs from PB maintain similar characteristics and have similar chondrogenic differentiation potential to those derived from BM, while producing comparable s-GAG expressions to chondrocytes.
    Matched MeSH terms: Chondrocytes/cytology*; Chondrocytes/physiology
  3. Mechanical compression controls the biosynthesis of human osteoarthritic chondrocytes in vitro
    Chong PP, Panjavarnam P, Ahmad WNHW, Chan CK, Abbas AA, Merican AM, et al.
    Clin Biomech (Bristol, Avon), 2020 10;79:105178.
    PMID: 32988676 DOI: 10.1016/j.clinbiomech.2020.105178
    BACKGROUND: Cartilage damage, which can potentially lead to osteoarthritis, is a leading cause of morbidity in the elderly population. Chondrocytes are sensitive to mechanical stimuli and their matrix-protein synthesis may be altered when chondrocytes experience a variety of in vivo loadings. Therefore, a study was conducted to evaluate the biosynthesis of isolated osteoarthritic chondrocytes which subjected to compression with varying dynamic compressive strains and loading durations.

    METHODS: The proximal tibia was resected as a single osteochondral unit during total knee replacement from patients (N = 10). The osteoarthritic chondrocytes were isolated from the osteochondral units, and characterized using reverse transcriptase-polymerase chain reaction. The isolated osteoarthritic chondrocytes were cultured and embedded in agarose, and then subjected to 10% and 20% uniaxial dynamic compression up to 8-days using a bioreactor. The morphological features and changes in the osteoarthritic chondrocytes upon compression were evaluated using scanning electron microscopy. Safranin O was used to detect the presence of cartilage matrix proteoglycan expression while quantitative analysis was conducted by measuring type VI collagen using an immunohistochemistry and fluorescence intensity assay.

    FINDINGS: Gene expression analysis indicated that the isolated osteoarthritic chondrocytes expressed chondrocyte-specific markers, including BGN, CD90 and HSPG-2. Moreover, the compressed osteoarthritic chondrocytes showed a more intense and broader deposition of proteoglycan and type VI collagen than control. The expression of type VI collagen was directly proportional to the duration of compression in which 8-days compression was significantly higher than 4-days compression. The 20% compression showed significantly higher intensity compared to 10% compression in 4- and 8-days.

    INTERPRETATION: The biosynthetic activity of human chondrocytes from osteoarthritic joints can be enhanced using selected compression regimes.

    Matched MeSH terms: Chondrocytes/metabolism; Chondrocytes/pathology*
  4. Total cell pooling in vitro: an effective isolation method for bone marrow-derived multipotent stromal cells
    Wee AS, Lim CK, Merican AM, Ahmad TS, Kamarul T
    In Vitro Cell Dev Biol Anim, 2013 Jun;49(6):424-32.
    PMID: 23708918 DOI: 10.1007/s11626-013-9626-0
    In vitro cellular proliferation and the ability to undergo multilineage differentiation make bone marrow-derived multipotent stromal cells (MSCs) potentially useful for clinical applications. Several methods have been described to isolate a homogenous bone marrow-derived MSCs population; however, none has been proven most effective, mainly due to their effects on proliferation and differentiation capability of the isolated cells. It is hypothesized that our newly established total cell pooling method may provide a better alternative as compared to the standard isolation method (density gradient centrifugation method). For the total cell pooling method, MSCs were isolated from rabbit bone marrow and were subsequently cultured in the growth medium without further separation as in the standard isolation method. The total cell pooling method was 65 min faster than the standard isolation method in completing cell isolation. Nevertheless, both methods did not differ significantly in the number of primary viable cells and population doubling time in the cultures (p > 0.05). The isolated cells from both methods expressed CD29 and CD44 markers, but not CD45 markers. Furthermore, they displayed multilineage differentiation characteristics of chondroblasts, osteoblasts, and adipocytes. In conclusion, both methods provide similar efficiency in the isolation of rabbit bone marrow-derived MSCs; however, the total cell pooling method is technically simpler and more cost effective than the standard isolation method.
    Matched MeSH terms: Chondrocytes/cytology
  5. The effect of TGF-beta1 and beta-estradiol on glycosaminoglycan and type II collagen distribution in articular chondrocyte cultures
    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: Chondrocytes/cytology; Chondrocytes/drug effects; Chondrocytes/metabolism*
  6. Fulltext Cell-based therapy for the treatment of focal articular cartilage lesions: a review of six years od studies in a Malaysian University Medical Centre
    Samsudin EZ, Kamarul T
    JUMMEC, 2014;17(2):1-11.
    MyJurnal
    Autologous chondrocyte implantation (ACI) is a significant technique that has gained widespread use for the treatment of focal articular cartilage damage. Since its inception in 2004, the Tissue Engineering Group (TEG) of the Faculty of Medicine, University Malaya has been dedicated to carrying out extensive research on this cell-based therapy. The objective of this report, comprising one clinical case report, six animal studies and one laboratory study, is to summarise and discuss TEG’s key findings. On the whole, we observed that the ACI technique was effective in regenerating hyaline-like cartilage in treated defects. Autologous chondrocytes and mesenchymal stem cells (MSC) were found to produce comparable tissue repair irrespective of the state of MSC differentiation, and the use of alginate-based scaffolding and oral pharmacotherapy (Glucosamine and Chondroitin Sulphate) was shown to enhance ACI-led tissue repair. ACI is suggested to be an efficient therapeutic option for the treatment of articular cartilage defects of the knee.
    Matched MeSH terms: Chondrocytes
  7. Fulltext Autologous chondrocyte implantation for knee focal cartilage defects: 3 years' follow-up at the University Malaya Medical Centre
    Abbas AA, Mohamad JA, Lydia AL, Selvaratnam L, Razif A, Ab-Rahim S, et al.
    JUMMEC, 2014;17(1):8-13.
    MyJurnal
    Autologous chondrocyte implantation (ACI) is a widely accepted procedure for the treatment of large, fullthickness chondral defects involving various joints, but its use in developing countries is limited because of high cost and failure rates due to limited resources and support systems. Five patients (age
    Matched MeSH terms: Chondrocytes
  8. Chondrocyte-alginate constructs with or without TGF-β1 produces superior extracellular matrix expression than monolayer cultures
    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: Chondrocytes/cytology*; Chondrocytes/drug effects; Chondrocytes/ultrastructure*
  9. Platelet-rich concentrate in serum-free medium enhances cartilage-specific extracellular matrix synthesis and reduces chondrocyte hypertrophy of human mesenchymal stromal cells encapsulated in alginate
    Samuel S, Ahmad RE, Ramasamy TS, Karunanithi P, Naveen SV, Kamarul T
    Platelets, 2019;30(1):66-74.
    PMID: 29090639 DOI: 10.1080/09537104.2017.1371287
    Platelet-rich concentrate (PRC), used in conjunction with other chondroinductive growth factors, have been shown to induce chondrogenesis of human mesenchymal stromal cells (hMSC) in pellet culture. However, pellet culture systems promote cell hypertrophy and the presence of other chondroinductive growth factors in the culture media used in previous studies obscures accurate determination of the effect of platelet itself in inducing chondrogenic differentiation. Hence, this study aimed to investigate the effect of PRC alone in enhancing the chondrogenic differentiation potential of human mesenchymal stromal cells (hMSC) encapsulated in three-dimensional alginate constructs. Cells encapsulated in alginate were cultured in serum-free medium supplemented with only 15% PRC. Scanning electron microscopy was used to determine the cell morphology. Chondrogenic molecular signature of hMSCs was determined by quantitative real-time PCR and verified at protein levels via immunohistochemistry and enzyme-linked immunosorbent assay. Results showed that the cells cultured in the presence of PRC for 24 days maintained a chondrocytic phenotype and demonstrated minimal upregulation of cartilaginous extracellular matrix (ECM) marker genes (SOX9, TNC, COL2, ACAN, COMP) and reduced expression of chondrocyte hypertrophy genes (Col X, Runx2) compared to the standard chondrogenic medium (p 
    Matched MeSH terms: Chondrocytes/metabolism*; Chondrocytes/pathology
  10. Protease activated receptor 2 and matriptase expression in the joints of cats with and without osteoarthritis
    Ariffin SMZ, Bennett D, Ferrell WR, Lockhart JC, Dunning L, Clements DN, et al.
    J Feline Med Surg, 2021 08;23(8):794-803.
    PMID: 33284033 DOI: 10.1177/1098612X20977796
    OBJECTIVES: The aim of this study was to determine the presence of protease-activated receptor 2 (PAR2) and matriptase proteins and quantify PAR2 and matriptase mRNA expression in the articular cartilage and synovial membrane of cats with and without osteoarthritis (OA).

    METHODS: A total of 28 articular cartilage samples from adult cats (14 OA and 14 normal), 10 synovial membranes from adult cats (five OA and five normal) and three cartilage samples from 9-week-old fetal cats were used. The presence of PAR2 and matriptase in the cartilage and synovial membrane of the adult samples was detected by immunohistochemical (IHC) staining, while real-time PCR was used for mRNA expression analyses in all samples.

    RESULTS: PAR2 was detected in all OA and normal articular cartilage and synovial membrane samples but confined to only a few superficial chondrocytes in the normal samples. Matriptase was only detected in OA articular cartilage and synovial membrane samples. PAR2 and matriptase mRNA expression were, however, detected in all cartilage and synovial membrane samples. PAR2 and matriptase mRNA expression levels in OA articular cartilage were five (P <0.001) and 3.3 (P <0.001) times higher than that of the healthy group, respectively. There was no significant difference (P = 0.05) in the OA synovial membrane PAR2 and matriptase mRNA expression compared with the normal samples.

    CONCLUSIONS AND RELEVANCE: Detection of PAR2 and matriptase proteins and gene expression in feline articular tissues is a novel and important finding, and supports the hypothesis that serine proteases are involved in the pathogenesis of feline OA. The consistent presence of PAR2 and matriptase protein in the cytoplasm of OA chondrocytes suggests a possible involvement of proteases in cartilage degradation. Further investigations into the PAR2 and matriptase pathobiology could enhance our understanding of the proteolytic cascades in feline OA, which might lead to the development of novel therapeutic strategies.

    Matched MeSH terms: Chondrocytes
  11. Effect of transforming growth factor-β3 on mono and multilayer chondrocytes
    Sefat F, Youseffi M, Khaghani SA, Soon CF, Javid F
    Cytokine, 2016 07;83:118-126.
    PMID: 27108397 DOI: 10.1016/j.cyto.2016.04.008
    Articular cartilage is an avascular and flexible connective tissue found in joints. It produces a cushioning effect at the joints and provides low friction to protect the ends of the bones from wear and tear/damage. It has poor repair capacity and any injury can result pain and loss of mobility. Transforming growth factor-beta (TGF-β), a cytokine superfamily, regulates cell function, including differentiation and proliferation. Although the function of the TGF-βs in various cell types has been investigated, their function in cartilage repair is as yet not fully understood. The effect of TGF-β3 in biological regulation of primary chondrocyte was investigated in this work. TGF-β3 provided fibroblastic morphology to chondrocytes and therefore overall reduction in cell proliferation was observed. The length of the cells supplemented with TGF-β3 were larger than the cells without TGF-β3 treatment. This was caused by the fibroblast like cells (dedifferentiated chondrocytes) which occupied larger areas compared to cells without TGF-β3 addition. The healing process of the model wound closure assay of chondrocyte multilayer was slowed down by TGF-β3, and this cytokine negatively affected the strength of chondrocyte adhesion to the cell culture surface.
    Matched MeSH terms: Chondrocytes/metabolism*; Chondrocytes/pathology
  12. Fulltext The role of vitamin E in preventing and treating osteoarthritis - a review of the current evidence
    Chin KY, Ima-Nirwana S
    Front Pharmacol, 2018;9:946.
    PMID: 30186176 DOI: 10.3389/fphar.2018.00946
    Osteoarthritis is a debilitating disease of the joint involving cartilage degeneration and chondrocytes apoptosis. Oxidative stress is one of the many proposed mechanisms underpinning joint degeneration in osteoarthritis. The current pharmacotherapies emphasize pain and symptomatic management of the patients but do not alter the biological processes underlying the cartilage degeneration. Vitamin E is a potential agent to prevent or treat osteoarthritis due to its antioxidant and anti-inflammatory effects. This review aims to summarize the current evidence on the relationship between vitamin E and osteoarthritis derived from preclinical and human studies. Cellular studies showed that vitamin E mitigated oxidative stress in cartilage explants or chondrocyte culture invoked by mechanical stress or free radicals. Animal studies suggested that vitamin E treatment prevented cartilage degeneration and improve oxidative status in animal models of osteoarthritis. Low circulating or synovial vitamin E was observed in human osteoarthritic patients compared to healthy controls. Observational studies also demonstrated that vitamin E was related to induction or progression of osteoarthritis in the general population. Vitamin E supplementation might improve the outcomes in patients with osteoarthritis, but negative results were also reported. Different isomers of vitamin E might possess distinct anti-osteoarthritic effects. As a conclusion, vitamin E may retard the progression of osteoarthritis by ameliorating oxidative stress and inflammation of the joint. Further studies are warranted to develop vitamin E as an anti-osteoarthritis agent to reduce the global burden of this disease.
    Matched MeSH terms: Chondrocytes
  13. The formation of human auricular cartilage from microtic tissue: An in vivo study
    Ishak MF, See GB, Hui CK, Abdullah Ab, Saim Lb, Saim Ab, et al.
    Int J Pediatr Otorhinolaryngol, 2015 Oct;79(10):1634-9.
    PMID: 26250439 DOI: 10.1016/j.ijporl.2015.06.034
    This study aimed to isolate, culture-expand and characterize the chondrocytes isolated from microtic cartilage and evaluate its potential as a cell source for ear cartilage reconstruction. Specific attention was to construct the auricular cartilage tissue by using fibrin as scaffold.
    Matched MeSH terms: Chondrocytes/cytology*; Chondrocytes/metabolism
  14. Fulltext Cartilage regeneration by chondrogenic induced adult stem cells in osteoarthritic sheep model
    Ude CC, Sulaiman SB, Min-Hwei N, Hui-Cheng C, Ahmad J, Yahaya NM, et al.
    PLoS One, 2014;9(6):e98770.
    PMID: 24911365 DOI: 10.1371/journal.pone.0098770
    In this study, Adipose stem cells (ADSC) and bone marrow stem cells (BMSC), multipotent adult cells with the potentials for cartilage regenerations were induced to chondrogenic lineage and used for cartilage regenerations in surgically induced osteoarthritis in sheep model.
    Matched MeSH terms: Chondrocytes/cytology; Chondrocytes/metabolism
  15. Fulltext Gelatin Microsphere for Cartilage Tissue Engineering: Current and Future Strategies
    Sulaiman SB, Idrus RBH, Hwei NM
    Polymers (Basel), 2020 Oct 19;12(10).
    PMID: 33086577 DOI: 10.3390/polym12102404
    The gelatin microsphere (GM) provides an attractive option for tissue engineering due to its versatility, as reported by various studies. This review presents the history, characteristics of, and the multiple approaches to, the production of GM, and in particular, the water in oil emulsification technique. Thereafter, the application of GM as a drug delivery system for cartilage diseases is introduced. The review then focusses on the emerging application of GM as a carrier for cells and biologics, and biologics delivery within a cartilage construct. The influence of GM on chondrocytes in terms of promoting chondrocyte proliferation and chondrogenic differentiation is highlighted. Furthermore, GM seeded with cells has been shown to have a high tendency to form aggregates; hence the concept of using GM seeded with cells as the building block for the formation of a complex tissue construct. Despite the advancement in GM research, some issues must still be addressed, particularly the improvement of GM's ability to home to defect sites. As such, the strategy of intraarticular injection of GM seeded with antibody-coated cells is proposed. By addressing this in future studies, a better-targeted delivery system, that would result in more effective intervention, can be achieved.
    Matched MeSH terms: Chondrocytes
  16. Extracellular matrix integrity affects the mechanical behaviour of in-situ chondrocytes under compression
    Moo EK, Han SK, Federico S, Sibole SC, Jinha A, Abu Osman NA, et al.
    J Biomech, 2014 Mar 21;47(5):1004-13.
    PMID: 24480705 DOI: 10.1016/j.jbiomech.2014.01.003
    Cartilage lesions change the microenvironment of cells and may accelerate cartilage degradation through catabolic responses from chondrocytes. In this study, we investigated the effects of structural integrity of the extracellular matrix (ECM) on chondrocytes by comparing the mechanics of cells surrounded by an intact ECM with cells close to a cartilage lesion using experimental and numerical methods. Experimentally, 15% nominal compression was applied to bovine cartilage tissues using a light-transmissible compression system. Target cells in the intact ECM and near lesions were imaged by dual-photon microscopy. Changes in cell morphology (N(cell)=32 for both ECM conditions) were quantified. A two-scale (tissue level and cell level) Finite Element (FE) model was also developed. A 15% nominal compression was applied to a non-linear, biphasic tissue model with the corresponding cell level models studied at different radial locations from the centre of the sample in the transient phase and at steady state. We studied the Green-Lagrange strains in the tissue and cells. Experimental and theoretical results indicated that cells near lesions deform less axially than chondrocytes in the intact ECM at steady state. However, cells near lesions experienced large tensile strains in the principal height direction, which are likely associated with non-uniform tissue radial bulging. Previous experiments showed that tensile strains of high magnitude cause an up-regulation of digestive enzyme gene expressions. Therefore, we propose that cartilage degradation near tissue lesions may be due to the large tensile strains in the principal height direction applied to cells, thus leading to an up-regulation of catabolic factors.
    Matched MeSH terms: Chondrocytes/physiology*
  17. Dual photon excitation microscopy and image threshold segmentation in live cell imaging during compression testing
    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: Chondrocytes/cytology; Chondrocytes/metabolism*
  18. Fulltext The properties of chondrocyte membrane reservoirs and their role in impact-induced cell death
    Moo EK, Amrein M, Epstein M, Duvall M, Abu Osman NA, Pingguan-Murphy B, et al.
    Biophys J, 2013 Oct 1;105(7):1590-600.
    PMID: 24094400 DOI: 10.1016/j.bpj.2013.08.035
    Impact loading of articular cartilage causes extensive chondrocyte death. Cell membranes have a limited elastic range of 3-4% strain but are protected from direct stretch during physiological loading by their membrane reservoir, an intricate pattern of membrane folds. Using a finite-element model, we suggested previously that access to the membrane reservoir is strain-rate-dependent and that during impact loading, the accessible membrane reservoir is drastically decreased, so that strains applied to chondrocytes are directly transferred to cell membranes, which fail when strains exceed 3-4%. However, experimental support for this proposal is lacking. The purpose of this study was to measure the accessible membrane reservoir size for different membrane strain rates using membrane tethering techniques with atomic force microscopy. We conducted atomic force spectroscopy on isolated chondrocytes (n = 87). A micron-sized cantilever was used to extract membrane tethers from cell surfaces at constant pulling rates. Membrane tethers could be identified as force plateaus in the resulting force-displacement curves. Six pulling rates were tested (1, 5, 10, 20, 40, and 80 μm/s). The size of the membrane reservoir, represented by the membrane tether surface areas, decreased exponentially with increasing pulling rates. The current results support our theoretical findings that chondrocytes exposed to impact loading die because of membrane ruptures caused by high tensile membrane strain rates.
    Matched MeSH terms: Chondrocytes/physiology; Chondrocytes/ultrastructure; Chondrocytes/chemistry*
  19. Stem cell genes are poorly expressed in chondrocytes from microtic cartilage
    Ishak MF, Chua KH, Asma A, Saim L, Aminuddin BS, Ruszymah BH, et al.
    Int J Pediatr Otorhinolaryngol, 2011 Jun;75(6):835-40.
    PMID: 21543123 DOI: 10.1016/j.ijporl.2011.03.021
    This study was aimed to see the difference between chondrocytes from normal cartilage compared to chondrocytes from microtic cartilage. Specific attentions were to characterize the growth of chondrocytes in terms of cell morphology, growth profile and RT-PCR analysis.
    Matched MeSH terms: Chondrocytes/cytology*; Chondrocytes/metabolism
  20. Growth kinetic study on normal and microtic chondrocytes of human auricular cartilage
    Ishak MF, Aminuddin BS, Asma A, Lokman BS, Ruszymah BH, Goh BS
    Med J Malaysia, 2008 Jul;63 Suppl A:117-8.
    PMID: 19025013
    Chondrocytes were isolated from normal and microtic human auricular cartilage after ear surgery carried out at Universiti Kebangsaan Malaysia Medical Centre. Chondrocytes were cultured and expanded until passage 4. After reached confluence, cultured chondrocytes at each passage (P1, P2, P3 and P4) were harvested and assigned for growth profile analysis. There was no significant difference in cell viability between both normal and microtic samples (p = 0.84). Both samples showed no significant differences for growth profile parameters in terms of growth rate, population doubling time and total number of cell doubling, except in passage 1, where there is significant difference in cell growth rate (p = 0.004). This preliminary data has indicated that chondrocytes from microtic cartilage has the potential to be used in the reconstruction of human pinna in the future.
    Matched MeSH terms: Chondrocytes/cytology*; Chondrocytes/physiology
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