Displaying publications 21 - 40 of 145 in total

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  1. Fulltext Uniaxial Cyclic Tensile Stretching at 8% Strain Exclusively Promotes Tenogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stromal Cells
    Nam HY, Pingguan-Murphy B, Abbas AA, Merican AM, Kamarul T
    Stem Cells Int, 2019;2019:9723025.
    PMID: 30918524 DOI: 10.1155/2019/9723025
    The present study was conducted to establish the amount of mechanical strain (uniaxial cyclic stretching) required to provide optimal tenogenic differentiation expression in human mesenchymal stromal cells (hMSCs) in vitro, in view of its potential application for tendon maintenance and regeneration. Methods. In the present study, hMSCs were subjected to 1 Hz uniaxial cyclic stretching for 6, 24, 48, and 72 hours; and were compared to unstretched cells. Changes in cell morphology were observed under light and atomic force microscopy. The tenogenic, osteogenic, adipogenic, and chondrogenic differentiation potential of hMSCs were evaluated using biochemical assays, extracellular matrix expressions, and selected mesenchyme gene expression markers; and were compared to primary tenocytes. Results. Cells subjected to loading displayed cytoskeletal coarsening, longer actin stress fiber, and higher cell stiffness as early as 6 hours. At 8% and 12% strains, an increase in collagen I, collagen III, fibronectin, and N-cadherin production was observed. Tenogenic gene expressions were highly expressed (p < 0.05) at 8% (highest) and 12%, both comparable to tenocytes. In contrast, the osteoblastic, chondrogenic, and adipogenic marker genes appeared to be downregulated. Conclusion. Our study suggests that mechanical loading at 8% strain and 1 Hz provides exclusive tenogenic differentiation; and produced comparable protein and gene expression to primary tenocytes.
  2. Fulltext Adipose and Bone Marrow Derived-Mesenchymal Stromal Cells Express Similar Tenogenic Expression Levels when Subjected to Mechanical Uniaxial Stretching In Vitro
    Nam HY, Draman Yusof MR, Kamarul T
    Stem Cells Int, 2023;2023:4907230.
    PMID: 36756494 DOI: 10.1155/2023/4907230
    The present study was conducted to determine whether adipose derived mesenchymal stromal cells (AD-MSCs) or bone marrow derived-MSCs (BM-MSCs) would provide superior tenogenic expressions when subjected to cyclical tensile loading. The results for this would indicate the best choice of MSCs source to be used for cell-based tendon repair strategies. Both AD-MSCs and BM-MSCs were obtained from ten adult donors (N = 10) and cultured in vitro. At passaged-2, cells from both groups were subjected to cyclical stretching at 1 Hz and 8% of strain. Cellular morphology, orientation, proliferation rate, protein, and gene expression levels were compared at 0, 24, and 48 hours of stretching. In both groups, mechanical stretching results in similar morphological changes, and the redirection of cell alignment is perpendicular to the direction of stretching. Loading at 8% strain did not significantly increase proliferation rates but caused an increase in total collagen expression and tenogenic gene expression levels. In both groups, these levels demonstrated no significant differences suggesting that in a similar loading environment, both cell types possess similar tenogenic potential. In conclusion, AD-MSCs and BM-MSCs both demonstrate similar tenogenic phenotypic and gene expression levels when subjected to cyclic tensile loading at 1 Hz and 8% strain, thus, suggesting that the use of either cell source may be suitable for tendon repair.
  3. Fulltext miR-524-5p of the primate-specific C19MC miRNA cluster targets TP53IPN1- and EMT-associated genes to regulate cellular reprogramming
    Nguyen PNN, Choo KB, Huang CJ, Sugii S, Cheong SK, Kamarul T
    Stem Cell Res Ther, 2017 09 29;8(1):214.
    PMID: 28962647 DOI: 10.1186/s13287-017-0666-3
    BACKGROUND: Introduction of the transcription factors Oct4, Sox2, Klf4, and c-Myc (OSKM) is able to 'reprogram' somatic cells to become induced pluripotent stem cells (iPSCs). Several microRNAs (miRNAs) are known to enhance reprogramming efficiency when co-expressed with the OSKM factors. The primate-specific chromosome 19 miRNA cluster (C19MC) is essential in primate reproduction, development, and differentiation. miR-524-5p, a C19MC member, is highly homologous to the reprogramming miR-520d-5p; we also reported that miR-524-5p was expressed in iPSCs but not mesenchymal stem cells (MSCs). This study aimed to elucidate possible contributions of miR-524-5p to the reprogramming process.

    METHODS: A miR-524-5p precursor was introduced into human fibroblast HFF-1 in the presence of OSKM, and the relative number of embryonic stem cell (ESC)-like colonies that stained positively with alkaline phosphatase (AP) and Nanog were quantified to determine reprogramming efficiency. A miR-524-5p mimic was transfected to MSCs to investigate the effects of miR-524-5p on TP53INP1, ZEB2, and SMAD4 expression by real-time polymerase chain reaction (PCR) and Western blot. Direct gene targeting was confirmed by luciferase activity. A phylogenetic tree of TP53INP1 was constructed by the Clustal method. Contribution of miR-524-5p to cell proliferation and apoptosis was examined by cell counts, BrdU, MTT, and cell death assays, and pluripotency gene expression by real-time PCR.

    RESULTS: Co-expressing the miR-524 precursor with OSKM resulted in a two-fold significant increase in the number of AP- and Nanog-positive ESC-like colonies, indicating a role for miR-524-5p in reprogramming. The putative target, TP53INP1, showed an inverse expression relationship with miR-524-5p; direct TP53INP1 targeting was confirmed in luciferase assays. miR-524-5p-induced TP53INP1 downregulation enhanced cell proliferation, suppressed apoptosis, and upregulated the expression of pluripotency genes, all of which are critical early events of the reprogramming process. Interestingly, the TP53INP1 gene may have co-evolved late with the primate-specific miR-524-5p. miR-524-5p also promoted mesenchymal-to-epithelial transition (MET), a required initial event of reprogramming, by directly targeting the epithelial-to-mesenchymal transition (EMT)-related genes, ZEB2 and SMAD4.

    CONCLUSIONS: Via targeting TP53INP1, ZEB2, and SMAD4, miR-524-5p contributes to the early stage of inducing pluripotency by promoting cell proliferation, inhibiting apoptosis, upregulating expression of pluripotency genes, and enhancing MET. Other C19MC miRNAs may have similar reprogramming functions.

  4. Fulltext Change in facial shape in two cohorts of Japanese adult female students twenty years apart
    Hossain MG, Saw A, Ohtsuki F, Lestrel PE, Kamarul T
    Singapore Med J, 2011 Nov;52(11):818-23.
    PMID: 22173252
    Secular changes in the head and body dimensions of the Japanese population were recorded during the last century, but studies on Japanese adult facial shape were poorly documented. This study aimed to document the secular changes in facial shape and their association with craniofacial measures of Japanese adult female students.
  5. Fulltext Multiple regression analysis of anthropometric measurements influencing the cephalic index of male Japanese university students
    Hossain MG, Saw A, Alam R, Ohtsuki F, Kamarul T
    Singapore Med J, 2013 Sep;54(9):516-20.
    PMID: 24068061
    INTRODUCTION: Cephalic index (CI), the ratio of head breadth to head length, is widely used to categorise human populations. The aim of this study was to access the impact of anthropometric measurements on the CI of male Japanese university students.

    METHODS: This study included 1,215 male university students from Tokyo and Kyoto, selected using convenient sampling. Multiple regression analysis was used to determine the effect of anthropometric measurements on CI.

    RESULTS: The variance inflation factor (VIF) showed no evidence of a multicollinearity problem among independent variables. The coefficients of the regression line demonstrated a significant positive relationship between CI and minimum frontal breadth (p < 0.01), bizygomatic breadth (p < 0.01) and head height (p < 0.05), and a negative relationship between CI and morphological facial height (p < 0.01) and head circumference (p < 0.01). Moreover, the coefficient and odds ratio of logistic regression analysis showed a greater likelihood for minimum frontal breadth (p < 0.01) and bizygomatic breadth (p < 0.01) to predict round-headedness, and morphological facial height (p < 0.05) and head circumference (p < 0.01) to predict long-headedness. Stepwise regression analysis revealed bizygomatic breadth, head circumference, minimum frontal breadth, head height and morphological facial height to be the best predictor craniofacial measurements with respect to CI.

    CONCLUSION: The results suggest that most of the variables considered in this study appear to influence the CI of adult male Japanese students.
  6. Fulltext Avoiding diagnostic pitfalls in mimics of neoplasia: the importance of a comprehensive diagnostic approach
    Samsudin EZ, Kamarul T, Mansor A
    Singapore Med J, 2015 May;56(5):e92-5.
    PMID: 26034328 DOI: 10.11622/smedj.2015082
    Any medical diagnosis should take a multimodal approach, especially those involving tumour-like conditions, as entities that mimic neoplasms have overlapping features and may present detrimental outcomes if they are underdiagnosed. These case reports present diagnostic pitfalls resulting from overdependence on a single diagnostic parameter for three musculoskeletal neoplasm mimics: brown tumour (BT) that was mistaken for giant cell tumour (GCT), methicillin-resistant Staphylococcus aureus osteomyelitis mistaken for osteosarcoma and a pseudoaneurysm mistaken for a soft tissue sarcoma. Literature reviews revealed five reports of BT simulating GCT, four reports of osteomyelitis mimicking osteosarcoma and five reports of a pseudoaneurysm imitating a soft tissue sarcoma. Our findings highlight the therapeutic dilemmas that arise with musculoskeletal mimics, as well as the importance of thorough investigation to distinguish mimickers from true neoplasms.
  7. Fulltext Attrition of Hepatic Damage Inflicted by Angiotensin II with α-Tocopherol and β-Carotene in Experimental Apolipoprotein E Knock-out Mice
    Gopal K, Gowtham M, Sachin S, Ravishankar Ram M, Shankar EM, Kamarul T
    Sci Rep, 2015 Dec 16;5:18300.
    PMID: 26670291 DOI: 10.1038/srep18300
    Angiotensin II is one of the key regulatory peptides implicated in the pathogenesis of liver disease. The mechanisms underlying the salubrious role of α-tocopherol and β-carotene on liver pathology have not been comprehensively assessed. Here, we investigated the mechanisms underlying the role of Angiotensin II on hepatic damage and if α-tocopherol and β-carotene supplementation attenuates hepatic damage. Hepatic damage was induced in Apoe(-/-)mice by infusion of Angiotensin II followed by oral administration with α-tocopherol and β-carotene-enriched diet for 60 days. Investigations showed fibrosis, kupffer cell hyperplasia, hepatocyte degeneration and hepatic cell apoptosis; sinusoidal dilatation along with haemorrhages; evidence of fluid accumulation; increased ROS level and increased AST and ALT activities. In addition, tPA and uPA were down-regulated due to 42-fold up-regulation of PAI-1. MMP-2, MMP-9, MMP-12, and M-CSF were down-regulated in Angiotensin II-treated animals. Notably, α-tocopherol and β-carotene treatment controlled ROS, fibrosis, hepatocyte degeneration, kupffer cell hyperplasia, hepatocyte apoptosis, sinusoidal dilatation and fluid accumulation in the liver sinusoids, and liver enzyme levels. In addition, PAI-1, tPA and uPA expressions were markedly controlled by β-carotene treatment. Thus, Angiotensin II markedly influenced hepatic damage possibly by restraining fibrinolytic system. We concluded that α-tocopherol and β-carotene treatment has salubrious role in repairing hepatic pathology.
  8. Fulltext Incorporation of Fucoidan in β-Tricalcium phosphate-Chitosan scaffold prompts the differentiation of human bone marrow stromal cells into osteogenic lineage
    Puvaneswary S, Raghavendran HB, Talebian S, Murali MR, A Mahmod S, Singh S, et al.
    Sci Rep, 2016;6:24202.
    PMID: 27068453 DOI: 10.1038/srep24202
    In our previous study, we reported the fabrication and characterization of a novel tricalcium phosphate-fucoidan-chitosan (TCP-Fu-Ch) biocomposite scaffold. However, the previous report did not show whether the biocomposite scaffold can exhibit osteogenic differentiation of human bone marrow stromal cells in osteogenic media and normal media supplemented with platelet-derived growth factor (PDGF-BB). On day 15, the release of osteocalcin, was significant in the TCP-Fu-Ch scaffold, when compared with that in the TCP-Ch scaffold, and the level of release was approximately 8 and 6 ng/ml in osteogenic and normal media supplemented with PDGF-BB, respectively. Scanning electron microscopy of the TCP-Fu-Ch scaffold demonstrated mineralization and apatite layer formation on day 14, while the addition of PDGF-BB also improved the osteogenic differentiation of the scaffold. An array of gene expression analysis demonstrated that TCP-Fu-Ch scaffold cultured in osteogenic and normal media supplemented with PDGF-BB showed significant improvement in the expression of collagen 1, Runt-related transcription factor 2, osteonectin, bone gamma-carboxyglutamate protein, alkaline phosphatase, and PPA2, but a decline in the expression of integrin. Altogether, the present study demonstrated that fucoidan-incorporated TCP-Ch scaffold could be used in the differentiation of bone marrow stromal cells and can be a potential candidate for the treatment of bone-related ailments through tissue engineering technology.
  9. Fulltext Fiber density of collagen grafts impacts rabbit urethral regeneration
    Larsson HM, Vythilingam G, Pinnagoda K, Vardar E, Engelhardt EM, Sothilingam S, et al.
    Sci Rep, 2018 07 03;8(1):10057.
    PMID: 29968749 DOI: 10.1038/s41598-018-27621-9
    There is a need for efficient and "off-the-shelf" grafts in urethral reconstructive surgery. Currently available surgical techniques require harvesting of grafts from autologous sites, with increased risk of surgical complications and added patient discomfort. Therefore, a cost-effective and cell-free graft with adequate regenerative potential has a great chance to be translated into clinical practice. Tubular cell-free collagen grafts were prepared by varying the collagen density and fiber distribution, thereby creating a polarized low fiber density collagen graft (LD-graft). A uniform, high fiber density collagen graft (HD-graft) was engineered as a control. These two grafts were implanted to bridge a 2 cm long iatrogenic urethral defect in a rabbit model. Histology revealed that rabbits implanted with the LD-graft had a better smooth muscle regeneration compared to the HD-graft. The overall functional outcome assessed by contrast voiding cystourethrography showed patency of the urethra in 90% for the LD-graft and in 66.6% for the HD-graft. Functional regeneration of the rabbit implanted with the LD-graft could further be demonstrated by successful mating, resulting in healthy offspring. In conclusion, cell-free low-density polarized collagen grafts show better urethral regeneration than high-density collagen grafts.
  10. Fulltext Enhancement of mesenchymal stem cell chondrogenesis with short-term low intensity pulsed electromagnetic fields
    Parate D, Franco-Obregón A, Fröhlich J, Beyer C, Abbas AA, Kamarul T, et al.
    Sci Rep, 2017 08 25;7(1):9421.
    PMID: 28842627 DOI: 10.1038/s41598-017-09892-w
    Pulse electromagnetic fields (PEMFs) have been shown to recruit calcium-signaling cascades common to chondrogenesis. Here we document the effects of specified PEMF parameters over mesenchymal stem cells (MSC) chondrogenic differentiation. MSCs undergoing chondrogenesis are preferentially responsive to an electromagnetic efficacy window defined by field amplitude, duration and frequency of exposure. Contrary to conventional practice of administering prolonged and repetitive exposures to PEMFs, optimal chondrogenic outcome is achieved in response to brief (10 minutes), low intensity (2 mT) exposure to 6 ms bursts of magnetic pulses, at 15 Hz, administered only once at the onset of chondrogenic induction. By contrast, repeated exposures diminished chondrogenic outcome and could be attributed to calcium entry after the initial induction. Transient receptor potential (TRP) channels appear to mediate these aspects of PEMF stimulation, serving as a conduit for extracellular calcium. Preventing calcium entry during the repeated PEMF exposure with the co-administration of EGTA or TRP channel antagonists precluded the inhibition of differentiation. This study highlights the intricacies of calcium homeostasis during early chondrogenesis and the constraints that are placed on PEMF-based therapeutic strategies aimed at promoting MSC chondrogenesis. The demonstrated efficacy of our optimized PEMF regimens has clear clinical implications for future regenerative strategies for cartilage.
  11. A comparison study of different physical treatments on cartilage matrix derived porous scaffolds for tissue engineering applications
    Moradi A, Pramanik S, Ataollahi F, Abdul Khalil A, Kamarul T, Pingguan-Murphy B
    Sci Technol Adv Mater, 2014 Dec;15(6):065001.
    PMID: 27877731
    Native cartilage matrix derived (CMD) scaffolds from various animal and human sources have drawn attention in cartilage tissue engineering due to the demonstrable presence of bioactive components. Different chemical and physical treatments have been employed to enhance the micro-architecture of CMD scaffolds. In this study we have assessed the typical effects of physical cross-linking methods, namely ultraviolet (UV) light, dehydrothermal (DHT) treatment, and combinations of them on bovine articular CMD porous scaffolds with three different matrix concentrations (5%, 15% and 30%) to assess the relative strengths of each treatment. Our findings suggest that UV and UV-DHT treatments on 15% CMD scaffolds can yield architecturally optimal scaffolds for cartilage tissue engineering.
  12. Comparison of in vitro developmental competence of cloned caprine embryos using donor karyoplasts from adult bone marrow mesenchymal stem cells vs ear fibroblast cells
    Kwong PJ, Nam HY, Wan Khadijah WE, Kamarul T, Abdullah RB
    Reprod. Domest. Anim., 2014 Apr;49(2):249-53.
    PMID: 24456113 DOI: 10.1111/rda.12262
    The aim of this study was to produce cloned caprine embryos using either caprine bone marrow-derived mesenchymal stem cells (MSCs) or ear fibroblast cells (EFCs) as donor karyoplasts. Caprine MSCs were isolated from male Boer goats of an average age of 1.5 years. To determine the pluripotency of MSCs, the cells were induced to differentiate into osteocytes, chondrocytes and adipocytes. Subsequently, MSCs were characterized through cell surface antigen profiles using specific markers, prior to their use as donor karyoplasts for nuclear transfer. No significant difference (p > 0.05) in fusion rates was observed between MSCs (87.7%) and EFCs (91.3%) used as donor karyoplasts. The cleavage rate of cloned embryos derived with MSCs (87.0%) was similar (p > 0.05) to those cloned using EFCs (84.4%). However, the in vitro development of MSCs-derived cloned embryos (25.3%) to the blastocyst stage was significantly higher (p < 0.05) than those derived with EFCs (20.6%). In conclusion, MSCs could be reprogrammed by caprine oocytes, and production of cloned caprine embryos with MSCs improved their in vitro developmental competence, but not in their fusion and cleavage rate as compared to cloning using somatic cells such as EFCs.
  13. Proliferation and osteogenic differentiation of mesenchymal stromal cells in a novel porous hydroxyapatite scaffold
    Krishnamurithy G, Murali MR, Hamdi M, Abbas AA, Raghavendran HB, Kamarul T
    Regen Med, 2015;10(5):579-90.
    PMID: 26237702 DOI: 10.2217/rme.15.27
    To compare the effect of bovine bone derived porous hydroxyapatite (BDHA) scaffold on proliferation and osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells (hMSCs) compared with commercial hydroxyapatite (CHA) scaffold.
  14. Stability of cervical spine after one-level corpectomy using different numbers of screws and plate systems
    Rosli R, Abdul Kadir MR, Kamarul T
    Proc Inst Mech Eng H, 2014 Apr;228(4):342-9.
    PMID: 24622982 DOI: 10.1177/0954411914527074
    Anterior corpectomy and reconstruction using a plate with locking screws are standard procedures for the treatment of cervical spondylotic myelopathy. Although adding more screws to the construct will normally result in improved fixation stability, several issues need to be considered. Past reports have suggested that increasing the number of screws can result in the increase in spinal rigidity, decreased spine mobility, loss of bone and, possibly, screw loosening. In order to overcome this, options to have constrained, semi-constrained or hybrid screw and plate systems were later introduced. The purpose of this study is to compare the stability achieved by four and two screws using different plate systems after one-level corpectomy with placement of cage. A three-dimensional finite-element model of an intact C1-C7 segment was developed from computer tomography data sets, including the cortical bone, soft tissue and simulated corpectomy fusion at C4-C5. A spinal cage and an anterior cervical plate with different numbers of screws and plate systems were constructed to a fit one-level corpectomy of C5. Moment load of 1.0 N m was applied to the superior surface of C1, with C7 was fixed in all degrees of freedom. The kinematic stability of a two-screw plate was found to be statistically equivalent to a four-screw plate for one-level corpectomy. Thus, it can be a better option of fusion and infers comparable stability after one-level anterior cervical corpectomy, instead of a four-screw plate.
  15. Calcium-Silicate-Incorporated Gellan-Chitosan Induced Osteogenic Differentiation in Mesenchymal Stromal Cells
    Genasan K, Mehrali M, Veerappan T, Talebian S, Malliga Raman M, Singh S, et al.
    Polymers (Basel), 2021 Sep 22;13(19).
    PMID: 34641027 DOI: 10.3390/polym13193211
    Gellan-chitosan (GC) incorporated with CS: 0% (GC-0 CS), 10% (GC-10 CS), 20% (GC-20 CS) or 40% (GC-40 CS) w/w was prepared using freeze-drying method to investigate its physicochemical, biocompatible, and osteoinductive properties in human bone-marrow mesenchymal stromal cells (hBMSCs). The composition of different groups was reflected in physicochemical analyses performed using BET, FTIR, and XRD. The SEM micrographs revealed excellent hBMSCs attachment in GC-40 CS. The Alamar Blue assay indicated an increased proliferation and viability of seeded hBMSCs in all groups on day 21 as compared with day 0. The hBMSCs seeded in GC-40 CS indicated osteogenic differentiation based on an amplified alkaline-phosphatase release on day 7 and 14 as compared with day 0. These cells supported bone mineralization on GC-40 CS based on Alizarin-Red assay on day 21 as compared with day 7 and increased their osteogenic gene expression (RUNX2, ALP, BGLAP, BMP, and Osteonectin) on day 21. The GC-40 CS-seeded hBMSCs initiated their osteogenic differentiation on day 7 as compared with counterparts based on an increased expression of type-1 collagen and BMP2 in immunocytochemistry analysis. In conclusion, the incorporation of 40% (w/w) calcium silicate in gellan-chitosan showed osteoinduction potential in hBMSCs, making it a potential biomaterial to treat critical bone defects.
  16. Fulltext A comparative study on in vitro osteogenic priming potential of electron spun scaffold PLLA/HA/Col, PLLA/HA, and PLLA/Col for tissue engineering application
    Balaji Raghavendran HR, Puvaneswary S, Talebian S, Murali MR, Raman Murali M, Naveen SV, et al.
    PLoS One, 2014;9(8):e104389.
    PMID: 25140798 DOI: 10.1371/journal.pone.0104389
    A comparative study on the in vitro osteogenic potential of electrospun poly-L-lactide/hydroxyapatite/collagen (PLLA/HA/Col, PLLA/HA, and PLLA/Col) scaffolds was conducted. The morphology, chemical composition, and surface roughness of the fibrous scaffolds were examined. Furthermore, cell attachment, distribution, morphology, mineralization, extracellular matrix protein localization, and gene expression of human mesenchymal stromal cells (hMSCs) differentiated on the fibrous scaffolds PLLA/Col/HA, PLLA/Col, and PLLA/HA were also analyzed. The electrospun scaffolds with a diameter of 200-950 nm demonstrated well-formed interconnected fibrous network structure, which supported the growth of hMSCs. When compared with PLLA/H%A and PLLA/Col scaffolds, PLLA/Col/HA scaffolds presented a higher density of viable cells and significant upregulation of genes associated with osteogenic lineage, which were achieved without the use of specific medium or growth factors. These results were supported by the elevated levels of calcium, osteocalcin, and mineralization (P<0.05) observed at different time points (0, 7, 14, and 21 days). Furthermore, electron microscopic observations and fibronectin localization revealed that PLLA/Col/HA scaffolds exhibited superior osteoinductivity, when compared with PLLA/Col or PLLA/HA scaffolds. These findings indicated that the fibrous structure and synergistic action of Col and nano-HA with high-molecular-weight PLLA played a vital role in inducing osteogenic differentiation of hMSCs. The data obtained in this study demonstrated that the developed fibrous PLLA/Col/HA biocomposite scaffold may be supportive for stem cell based therapies for bone repair, when compared with the other two scaffolds.
  17. Fulltext Identification of Pathways Mediating Growth Differentiation Factor5-Induced Tenogenic Differentiation in Human Bone Marrow Stromal Cells
    Tan SL, Ahmad TS, Ng WM, Azlina AA, Azhar MM, Selvaratnam L, et al.
    PLoS One, 2015;10(11):e0140869.
    PMID: 26528540 DOI: 10.1371/journal.pone.0140869
    To date, the molecular signalling mechanisms which regulate growth factors-induced MSCs tenogenic differentiation remain largely unknown. Therefore, a study to determine the global gene expression profile of tenogenic differentiation in human bone marrow stromal cells (hMSCs) using growth differentiation factor 5 (GDF5) was conducted. Microarray analyses were conducted on hMSCs cultures supplemented with 100 ng/ml of GDF5 and compared to undifferentiated hMSCs and adult tenocytes. Results of QuantiGene® Plex assay support the use and interpretation of the inferred gene expression profiles and pathways information. From the 27,216 genes assessed, 873 genes (3.21% of the overall human transcriptome) were significantly altered during the tenogenic differentiation process (corrected p<0.05). The genes identified as potentially associated with tenogenic differentiation were ARHGAP29, CCL2, integrin alpha 8 and neurofilament medium polypeptides. These genes, were mainly associated with cytoskeleton reorganization (stress fibers formation) signaling. Pathway analysis demonstrated the potential molecular pathways involved in tenogenic differentiation were: cytoskeleton reorganization related i.e. keratin filament signaling and activin A signaling; cell adhesion related i.e. chemokine and adhesion signaling; and extracellular matrix related i.e. arachidonic acid production signaling. Further investigation using atomic force microscopy and confocal laser scanning microscopy demonstrated apparent cytoskeleton reorganization in GDF5-induced hMSCs suggesting that cytoskeleton reorganization signaling is an important event involved in tenogenic differentiation. Besides, a reduced nucleostemin expression observed suggested a lower cell proliferation rate in hMSCs undergoing tenogenic differentiation. Understanding and elucidating the tenogenic differentiation signalling pathways are important for future optimization of tenogenic hMSCs for functional tendon cell-based therapy and tissue engineering.
  18. Fulltext Fate of tenogenic differentiation potential of human bone marrow stromal cells by uniaxial stretching affected by stretch-activated calcium channel agonist gadolinium
    Nam HY, Balaji Raghavendran HR, Pingguan-Murphy B, Abbas AA, Merican AM, Kamarul T
    PLoS One, 2017;12(6):e0178117.
    PMID: 28654695 DOI: 10.1371/journal.pone.0178117
    The role for mechanical stimulation in the control of cell fate has been previously proposed, suggesting that there may be a role of mechanical conditioning in directing mesenchymal stromal cells (MSCs) towards specific lineage for tissue engineering applications. Although previous studies have reported that calcium signalling is involved in regulating many cellular processes in many cell types, its role in managing cellular responses to tensile loading (mechanotransduction) of MSCs has not been fully elucidated. In order to establish this, we disrupted calcium signalling by blocking stretch-activated calcium channel (SACC) in human MSCs (hMSCs) in vitro. Passaged-2 hMSCs were exposed to cyclic tensile loading (1 Hz + 8% for 6, 24, 48, and 72 hours) in the presence of the SACC blocker, gadolinium. Analyses include image observations of immunochemistry and immunofluorescence staining from extracellular matrix (ECM) production, and measuring related tenogenic and apoptosis gene marker expression. Uniaxial tensile loading increased the expression of tenogenic markers and ECM production. However, exposure to strain in the presence of 20 μM gadolinium reduced the induction of almost all tenogenic markers and ECM staining, suggesting that SACC acts as a mechanosensor in strain-induced hMSC tenogenic differentiation process. Although cell death was observed in prolonged stretching, it did not appear to be apoptosis mediated. In conclusion, the knowledge gained in this study by elucidating the role of calcium in MSC mechanotransduction processes, and that in prolonged stretching results in non-apoptosis mediated cell death may be potential useful for regenerative medicine applications.
  19. Fulltext The physicochemical and biomechanical profile of forsterite and its osteogenic potential of mesenchymal stromal cells
    Krishnamurithy G, Mohan S, Yahya NA, Mansor A, Murali MR, Raghavendran HRB, et al.
    PLoS One, 2019;14(3):e0214212.
    PMID: 30917166 DOI: 10.1371/journal.pone.0214212
    It has been demonstrated that nanocrystalline forsterite powder synthesised using urea as a fuel in sol-gel combustion method had produced a pure forsterite (FU) and possessed superior bioactive characteristics such as bone apatite formation and antibacterial properties. In the present study, 3D-scaffold was fabricated using nanocrystalline forsterite powder in polymer sponge method. The FU scaffold was used in investigating the physicochemical, biomechanics, cell attachment, in vitro biocompatibility and osteogenic differentiation properties. For physicochemical characterisation, Fourier-transform infrared spectroscopy (FTIR), Energy dispersive X-ray (EDX), X-ray diffraction (XRD), Raman spectroscopy, X-ray photoemission spectrometer (XPS) and Brunauer-Emmett-Teller (BET) were used. FTIR, EDX, XRD peaks and Raman spectroscopy demonstrated correlating to FU. The XPS confirmed the surface chemistry associating to FU. The BET revealed FU scaffold surface area of 12.67 m2/g and total pore size of 0.03 cm3/g. Compressive strength of the FU scaffold was found to be 27.18 ± 13.4 MPa. The human bone marrow derived mesenchymal stromal cells (hBMSCs) characterisation prior to perform seeding on FU scaffold verified the stromal cell phenotypic and lineage commitments. SEM, confocal images and presto blue viability assay suggested good cell attachment and proliferation of hBMSCs on FU scaffold and comparable to a commercial bone substitutes (cBS). Osteogenic proteins and gene expression from day 7 onward indicated FU scaffold had a significant osteogenic potential (p<0.05), when compared with day 1 as well as between FU and cBS. These findings suggest that FU scaffold has a greater potential for use in orthopaedic and/or orthodontic applications.
  20. 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 
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