Displaying all 20 publications

  1. Tan, S.L., Selvaratnam, L., Ahmad, T.S.
    JUMMEC, 2015;18(2):1-14.
    Tendon is a dense connective tissue that connects muscle to bone. Tendon can adapt to mechanical forces passing across it, through a reciprocal relationship between its cellular components (tenocytes and tenoblasts) and the extracellular matrix (ECM). In early development, the formation of scleraxis-expressing tendon progenitor population in the sclerotome is induced by a fibroblast growth factor signal secreted by the myotome. Tendon injury has been defined as a loss of cells or ECM caused by trauma. It represents a failure of cells and matrix adaptation to mechanical loading. Injury initiates attempts of tendon to repair itself, which has been defined as replacement of damaged or lost cells and ECM by new cells or new matrices. Tendon healing generally consists of four different phases: the inflammatory, proliferation, differentiation and remodelling phases. Clinically, tendons are repaired with a variety of surgical techniques, which show various degrees of success. In order to improve the conventional tendon repair methods, current tendon tissue engineering aims to investigate a repair method which can restore tissue defects with living cells, or cell based therapy. Advances in tissue engineering techniques would potentially yield to a cell-based product that could regenerate functional tendon tissue.
  2. 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.
  3. Boo, L., Sofiah, S., Selvaratnam, L., Tai, C.C., Pingguan-Murphy, B., Kamarul, T.
    Malays Orthop J, 2009;3(2):16-23.
    Purpose:To investigate the feasibilty of using processed human amniotic membrane (HAM) to support the attachment and proliferation of chondrocytes in vitro which it turn can be utilised as a cell delivery vehicle in tissue engineering applications. Methods: Fresh HAM obtained from patients undergoing routine elective ceasarean sections was harvested., processed and dried using either freez drying (FD) or air drying (AD) methods prior to sterilisation by gamma irradiation. Isolated, processed and characterised rabbit autologous chondrolytes were seeded on processsed HAM and cultured for up to three weeks. Cell attachment and proliferation were examined qualitatively using inverted brightfield microcospy. Results: Processed HAM appeared to allow cell attachment when implanted with chrondocytes. Although cells seeded on AD and FD HAM did not appear to attach as strongly as those seeded on glycerol preserved intact human amniotic membrane, these cells to be proliferated in cell culture conditions. Conclusion: Prelimanary results show that processed HAM chondrocyte attachment and proliferation.
  4. Abbas AA, Mohamad JA, Lydia AL, Selvaratnam L, Razif A, Ab-Rahim S, et al.
    JUMMEC, 2014;17(1):8-13.
    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
  5. Kamarul T, Ab-Rahim S, Tumin M, Selvaratnam L, Ahmad TS
    Eur Cell Mater, 2011 Mar 15;21:259-71; discussion 270-1.
    PMID: 21409755
    The effects of Glucosamine Sulphate (GS) and Chondroitin Sulphate (CS) on the healing of damaged and repaired articular cartilage were investigated. This study was conducted using 18 New Zealand white rabbits as experimental models. Focal cartilage defects, surgically created in the medial femoral condyle, were either treated by means of autologous chondrocyte implantation (ACI) or left untreated as controls. Rabbits were then divided into groups which received either GS+/-CS or no pharmacotherapy. Three rabbits from each group were sacrificed at 12 and 24 weeks post-surgery. Knees dissected from rabbits were then evaluated using gross quantification of repair tissue, glycosaminoglycan (GAG) assays, immunoassays and histological assessments. It was observed that, in contrast to untreated sites, surfaces of the ACI-repaired sites appeared smooth and continuous with the surrounding native cartilage. Histological examination demonstrated a typical hyaline cartilage structure; with proteoglycans, type II collagen and GAGs being highly expressed in repair areas. The improved regeneration of these repair sites was also noted to be significant over time (6 months vs. 3 months) and in GS and GS+CS groups compared to the untreated (without pharmacotherapy) group. Combination of ACI and pharmacotherapy (with glucosamine sulphate alone/ or with chondroitin sulphate) may prove beneficial for healing of damaged cartilage, particularly in relation to focal cartilage defects.
  6. Boo L, Selvaratnam L, Tai CC, Ahmad TS, Kamarul T
    J Mater Sci Mater Med, 2011 May;22(5):1343-56.
    PMID: 21461701 DOI: 10.1007/s10856-011-4294-7
    The use of mesenchymal stem cells (MSCs) in tissue repair and regeneration despite their multipotentiality has been limited by their cell source quantity and decelerating proliferative yield efficiency. A study was thus undertaken to determine the feasibility of using microcarrier beads in spinner flask cultures for MSCs expansion and compared to that of conventional monolayer cultures and static microcarrier cultures. Isolation and characterization of bone marrow derived MSCs were conducted from six adult New Zealand white rabbits. Analysis of cell morphology on microcarriers and culture plates at different time points (D0, D3, D10, D14) during cell culture were performed using scanning electron microscopy and bright field microscopy. Cell proliferation rates and cell number were measured over a period of 14 days, respectively followed by post-expansion characterization. MTT proliferation assay demonstrated a 3.20 fold increase in cell proliferation rates in MSCs cultured on microcarriers in spinner flask as compared to monolayer cultures (p < 0.05). Cell counts at day 14 were higher in those seeded on stirred microcarrier cultures (6.24 ± 0.0420 cells/ml) × 10(5) as compared to monolayer cultures (0.22 ± 0.004 cells/ml) × 10(5) and static microcarrier cultures (0.20 ± 0.002 cells/ml) × 10(5). Scanning electron microscopy demonstrated an increase in cell colonization of the cells on the microcarriers in stirred cultures. Bead-expanded MSCs were successfully differentiated into osteogenic and chondrogenic lineages. This system offers an improved and efficient alternative for culturing MSCs with preservation to their phenotype and multipotentiality.
  7. 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.
  8. 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.
  9. Chong PP, Selvaratnam L, Abbas AA, Kamarul T
    Open Life Sci, 2018 Jan;13:279-284.
    PMID: 33817094 DOI: 10.1515/biol-2018-0034
    Most studies highlight mesenchymal stem cells (MSCs) extracted primarily from bone marrow (BM), very few report the use of peripheral blood (PB), often due to the associated low seeding density and difficulties with extraction techniques. As ageing populations are becoming more predominant globally, together with escalating demands for MSC transplantation and tissue regeneration, obtaining quality MSCs suitable for induced differentiation and biological therapies becomes increasingly important. In this study, BM and PB were obtained from elderly patients and extracted MSCs grown in vitro to determine their successful isolation and expansion. Patients' socio-demographic background and other medical information were obtained from medical records. Successful and failed cultures were correlated with key demographic and medical parameters. A total of 112 samples (BM or PB) were used for this study. Of these, 50 samples (44.6%) were successfully cultured according to standardised criteria with no signs of contamination. Our comparative analyses demonstrated no statistical correlation between successful MSC cultures and any of the six demographic or medical parameters examined, including sample quantity, age, sex, race, habits and underlying comorbidities of sample donors. In conclusion, the present study demonstrates that typical demographics and comorbidities do not influence successful MSC isolation and expansion in culture.
  10. Roslan Z, Muhamad M, Selvaratnam L, Ab-Rahim S
    J Oncol, 2019;2019:4536302.
    PMID: 31031810 DOI: 10.1155/2019/4536302
    Low-density lipoprotein receptor (LDLR) has been an object of research since the 1970s because of its role in various cell functions. The LDLR family members include LRP5, LRP6, and LRP8. Even though LRP5, 6, and 8 are in the same family, intriguingly, these three proteins have various roles in physiological events, as well as in regulating different mechanisms in various kinds of cancers. LRP5, LRP6, and LRP8 have been shown to play important roles in a broad panel of cancers. LRP5 is highly expressed in many tissues and is involved in the modulation of glucose-induced insulin secretion, bone development, and cholesterol metabolism, as well as cancer progression. Recently, LRP5 has also been shown to play a role in chondroblastic subtype of osteosarcoma (OS) and prostate cancer and also in noncancer case such as osteoporosis. LRP6, which has been previously discovered to share the same structures as LRP5, has also been associated with many cancer progressions such as human triple negative breast cancer (TNBC), primary chronic lymphocytic leukemia (CLL), nonsmall cell lung cancer (NSCL), lung squamous cell carcinoma (LSCC), and hepatocellular carcinoma (HCC). In addition to its role in cancer progression, LRP8 (apolipoprotein E receptor 2 [APOER2]) has also been demonstrated to regulate canonical Wnt/β-catenin signaling pathway whereby this pathway plays a role in cell migration and development. Therefore, this review aimed to elucidate the role of LRP 5, 6, and 8 in regulating the cancer progression.
  11. Tan SL, Ahmad TS, Selvaratnam L, Kamarul T
    J Anat, 2013 Apr;222(4):437-50.
    PMID: 23510053 DOI: 10.1111/joa.12032
    Mesenchymal stem cells (MSCs) are recognized by their plastic adherent ability, fibroblastic-like appearance, expression of specific surface protein markers, and are defined by their ability to undergo multi-lineage differentiation. Although rabbit bone marrow-derived MSCs (rbMSCs) have been used extensively in previous studies especially in translational research, these cells have neither been defined morphologically and ultrastructurally, nor been compared with their counterparts in humans in their multi-lineage differentiation ability. A study was therefore conducted to define the morphology, surface marker proteins, ultrastructure and multi-lineage differentiation ability of rbMSCs. Herein, the primary rbMSC cultures of three adult New Zealand white rabbits (at least 4 months old) were used for three independent experiments. rbMSCs were isolated using the gradient-centrifugation method, an established technique for human MSCs (hMSCs) isolation. Cells were characterized by phase contrast microscopy observation, transmission electron microscopy analysis, reverse transcriptase-polymerase chain reaction (PCR) analysis, immunocytochemistry staining, flow cytometry, alamarBlue(®) assay, histological staining and quantitative (q)PCR analysis. The isolated plastic adherent cells were in fibroblastic spindle-shape and possessed eccentric, irregular-shaped nuclei as well as rich inner cytoplasmic zones similar to that of hMSCs. The rbMSCs expressed CD29, CD44, CD73, CD81, CD90 and CD166, but were negative (or dim positive) for CD34, CD45, CD117 and HLD-DR. Despite having similar morphology and phenotypic expression, rbMSCs possessed significantly larger cell size but had a lower proliferation rate as compared with hMSCs. Using established protocols to differentiate hMSCs, rbMSCs underwent osteogenic, adipogenic and chondrogenic differentiation. Interestingly, differentiated rbMSCs demonstrated higher levels of osteogenic (Runx2) and chondrogenic (Sox9) gene expressions than that of hMSCs (P  0.05). rbMSCs possess similar morphological characteristics to hMSCs, but have a higher potential for osteogenic and chondrogenic differentiation, despite having a lower cell proliferation rate than hMSCs. The characteristics reported here may be used as a comprehensive set of criteria to define or characterize rbMSCs.
  12. 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.
  13. Dashtdar H, Murali MR, Abbas AA, Suhaeb AM, Selvaratnam L, Tay LX, et al.
    Knee Surg Sports Traumatol Arthrosc, 2015 May;23(5):1368-77.
    PMID: 24146054 DOI: 10.1007/s00167-013-2723-5
    PURPOSE: To investigate whether mesenchymal stem cells (MSCs) seeded in novel polyvinyl alcohol (PVA)-chitosan composite hydrogel can provide comparable or even further improve cartilage repair outcomes as compared to previously established alginate-transplanted models.

    METHODS: Medial femoral condyle defect was created in both knees of twenty-four mature New Zealand white rabbits, and the animals were divided into four groups containing six animals each. After 3 weeks, the right knees were transplanted with PVA-chitosan-MSC, PVA-chitosan scaffold alone, alginate-MSC construct or alginate alone. The left knee was kept as untreated control. Animals were killed at the end of 6 months after transplantation, and the cartilage repair was assessed through Brittberg morphological score, histological grading by O'Driscoll score and quantitative glycosaminoglycan analysis.

    RESULTS: Morphological and histological analyses showed significant (p < 0.05) tissue repair when treated with PVA-chitosan-MSC or alginate MSC as compared to the scaffold only and untreated control. In addition, safranin O staining and the glycosaminoglycan (GAG) content were significantly higher (p < 0.05) in MSC treatment groups than in scaffold-only or untreated control group. No significant difference was observed between the PVA-chitosan-MSC- and alginate-MSC-treated groups.

    CONCLUSION: PVA-chitosan hydrogel seeded with mesenchymal stem cells provides comparable treatment outcomes to that of previously established alginate-MSC construct implantation. This study supports the potential use of PVA-chitosan hydrogel seeded with MSCs for clinical use in cartilage repair such as traumatic injuries.

  14. Tay LX, Ahmad RE, Dashtdar H, Tay KW, Masjuddin T, Ab-Rahim S, et al.
    Am J Sports Med, 2012 Jan;40(1):83-90.
    PMID: 21917609 DOI: 10.1177/0363546511420819
    Mesenchymal stem cells (MSCs) represent a promising alternative form of cell-based therapy for cartilage injury. However, the capacity of MSCs for chondrogenesis has not been fully explored. In particular, there is presently a lack of studies comparing the effectiveness of MSCs to conventional autologous chondrocyte (autoC) treatment for regeneration of full-thickness cartilage defects in vivo.
  15. Lee SY, Pereira BP, Yusof N, Selvaratnam L, Yu Z, Abbas AA, et al.
    Acta Biomater, 2009 Jul;5(6):1919-25.
    PMID: 19289306 DOI: 10.1016/j.actbio.2009.02.014
    A poly(vinyl alcohol) (PVA) hydrogel composite scaffold containing N,O-carboxymethylated chitosan (NOCC) was tested to assess its potential as a scaffold for cartilage tissue engineering in a weight-bearing environment. The mechanical properties under unconfined compression for different hydration periods were investigated. The effect of supplementing PVA with NOCC (20wt.% PVA:5vol.% NOCC) produced a porosity of 43.3% and this was compared against a non-porous PVA hydrogel (20g PVA: 100ml of water, control). Under non-hydrated conditions, the porous PVA-NOCC hydrogel behaved in a similar way to the control non-porous PVA hydrogel, with similar non-linear stress-strain response under unconfined compression (0-30% strain). After 7days' hydration, the porous hydrogel demonstrated a reduced stiffness (0.002kPa, at 25% strain), resulting in a more linear stiffness relationship over a range of 0-30% strain. Poisson's ratio for the hydrated non-porous and porous hydrogels ranged between 0.73 and 1.18, and 0.76 and 1.33, respectively, suggesting a greater fluid flow when loaded. The stress relaxation function for the porous hydrogel was affected by the hydration period (from 0 to 600s); however the percentage stress relaxation regained by about 95%, after 1200s for all hydration periods assessed. No significant differences were found between the different hydration periods between the porous hydrogels and control. The calculated aggregate modulus, H(A), for the porous hydrogel reduced drastically from 10.99kPa in its non-hydrated state to about 0.001kPa after 7days' hydration, with the calculated shear modulus reducing from 30.92 to 0.14kPa, respectively. The porous PVA-NOCC hydrogel conformed to a biphasic, viscoelastic model, which has the desired properties required for any scaffold in cartilage tissue engineering.
  16. Tan SL, Sulaiman S, Pingguan-Murphy B, Selvaratnam L, Tai CC, Kamarul T
    Cell Tissue Bank, 2011 Feb;12(1):59-70.
    PMID: 19953328 DOI: 10.1007/s10561-009-9164-x
    This study investigates the feasibility of processed human amnion (HAM) as a substrate for chondrogenic differentiation of mesenchymal stem cells (MSCs). HAM preparations processed by air drying (AD) and freeze drying (FD) underwent histological examination and MSC seeding in chondrogenic medium for 15 days. Monolayer cultures were used as control for chondrogenic differentiation and HAMs without cell seeding were used as negative control. Qualitative observations were made using scanning electron microscopy analysis and quantitative analyses were based on the sulfated glycosaminoglycans (GAG) assays performed on day 1 and day 15. Histological examination of HAM substrates before seeding revealed a smooth surface in AD substrates, while the FD substrates exhibited a porous surface. Cell attachment to AD and FD substrates on day 15 was qualitatively comparable. GAG were significantly highly expressed in cells seeded on FD HAM substrates. This study indicates that processed HAM is a potentially valuable material as a cell-carrier for MSC differentiation.
  17. Selvaratnam L, Abd Rahim S, Kamarul T, Chan KY, Sureshan S, Penafort R, et al.
    Med J Malaysia, 2005 Jul;60 Suppl C:49-52.
    PMID: 16381284
    In view of poor regeneration potential of the articular cartilage, in-vitro engineering of cartilage tissue offers a promising option for progressive joint disease. This study aims to develop a biologically engineered articular cartilage for autologous transplantation. The initial work involved determination of chondrocyte yield and viability, and morphological analysis. Cartilage was harvested from the knee, hip and shoulder joints of adult New Zealand white rabbits and chondrocytes were isolated by enzymatic digestion of the extra-cellular matrix before serial cultivation in DMEM/Ham's F12 media as monolayer cultures. No differences were noted in cell yield. Although chondrocytes viability was optimal (>93%) following harvest from native cartilage, their viability tended to be lowered on passaging. Chondrocytes aggregated in isogenous colonies comprising ovoid cells with intimate intracellular contacts and readily exhibited Safranin-O positive matrix; features typically associated with articular cartilage in-vivo. However, chondrocytes also existed concurrently in scattered bipolar/multipolar forms lacking Safranin-O expression. Therefore, early data demonstrated successful serial culture of adult chondrocytes with differentiated morphology seen in established chondrocyte colonies synthesizing matrix proteoglycans.
  18. 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.
  19. Dashtdar H, Murali MR, Selvaratnam L, Balaji Raghavendran H, Suhaeb AM, Ahmad TS, et al.
    PeerJ, 2016;4:e1650.
    PMID: 26966647 DOI: 10.7717/peerj.1650
    Chondrogenic differentiation of mesenchymal stromal cells (MSCs) in the form of pellet culture and encapsulation in alginate beads has been widely used as conventional model for in vitro chondrogenesis. However, comparative characterization between differentiation, hypertrophic markers, cell adhesion molecule and ultrastructural changes during alginate and pellet culture has not been described. Hence, the present study was conducted comparing MSCs cultured in pellet and alginate beads with monolayer culture. qPCR was performed to assess the expression of chondrogenic, hypertrophic, and cell adhesion molecule genes, whereas transmission electron microscopy (TEM) was used to assess the ultrastructural changes. In addition, immunocytochemistry for Collagen type II and aggrecan and glycosaminoglycan (GAG) analysis were performed. Our results indicate that pellet and alginate bead cultures were necessary for chondrogenic differentiation of MSC. It also indicates that cultures using alginate bead demonstrated significantly higher (p < 0.05) chondrogenic but lower hypertrophic (p < 0.05) gene expressions as compared with pellet cultures. N-cadherin and N-CAM1 expression were up-regulated in second and third weeks of culture and were comparable between the alginate bead and pellet culture groups, respectively. TEM images demonstrated ultrastructural changes resembling cell death in pellet cultures. Our results indicate that using alginate beads, MSCs express higher chondrogenic but lower hypertrophic gene expression. Enhanced production of extracellular matrix and cell adhesion molecules was also observed in this group. These findings suggest that alginate bead culture may serve as a superior chondrogenic model, whereas pellet culture is more appropriate as a hypertrophic model of chondrogenesis.
  20. Lee SY, Wee AS, Lim CK, Abbas AA, Selvaratnam L, Merican AM, et al.
    J Mater Sci Mater Med, 2013 Jun;24(6):1561-70.
    PMID: 23512151 DOI: 10.1007/s10856-013-4907-4
    This study aims to pre-assess the in vitro and in vivo biocompatibility of poly(vinyl alcohol)-carboxylmethyl-chitosan-poly(ethylene glycol) (PCP) scaffold. PCP was lyophilised to create supermacroporous structures. 3-(4, 5-dimethyl-thiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and immunohistochemistry (IHC) were used to evaluate the effectiveness of PCP scaffolds for chondrocytes attachment and proliferation. The ultrastructural was assessed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Extracellular matrix (ECM) formation was evaluated using collagen type-II staining, glycosaminoglycan (GAG) and collagen assays. Histological analysis was conducted on 3-week implanted Sprague-Dawley rats. The MTT, IHC, SEM and TEM analyses confirm that PCP scaffolds promoted cell attachment and proliferation in vitro. The chondrocyte-PCP constructs secreted GAG and collagen type-II, both increased significantly from day-14 to day-28 (P 
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