Publications by authors named "Chin Keong Lim"

8 Publications

  • Page 1 of 1

Synergistic effects of chitosan scaffold and TGFβ1 on the proliferation and osteogenic differentiation of dental pulp stem cells derived from human exfoliated deciduous teeth.

Arch Oral Biol 2014 Dec 6;59(12):1400-11. Epub 2014 Sep 6.

Conservative Department, School of Dental Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia.

Objective: Multipotent stem cells derived from human exfoliated deciduous teeth (SHED) represent a promising cell source for tissue regeneration. In the present study we decided to test the inductive effect of chitosan and transforming growth factor-β1 (TGFβ1) as a scaffold/factor combination on SHED proliferation and osteogenic differentiation.

Design: Cell proliferation was quantitatively assessed by PrestoBlue, live/dead assay was performed and cell attachment to chitosan scaffold was examined by scanning electron microscopy (SEM). For osteogenic differentiation analysis, alkaline phosphatase activity was quantified, cells were stained with Alizarin Red, and the lineage specific genes/proteins ALP, COL I, BSP, and OCN were analysed by real-time PCR and Western blot.

Results: SHED remained viable and attached well to the chitosan structure. Moreover, TGFβ1 significantly enhanced the proliferative activity of SHED on the chitosan scaffold. Our data further revealed that chitosan and TGFβ1 enhanced the osteogenic differentiation of SHED, as evidenced by high ALP activity, strong mineral deposition, and the up-regulation of ALP, COL I, BSP, and OCN gene/protein expression.

Conclusion: Together, data from our study indicate that the combination of chitosan scaffolds and TGFβ1 enhanced proliferation and osteogenic differentiation of SHED. These findings suggest that the combined application of chitosan scaffold and TGFβ1 in conjunction with SHED might be beneficial for in vivo bone regeneration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.archoralbio.2014.08.015DOI Listing
December 2014

Physical properties and biocompatibility of oligochitosan membrane film as wound dressing.

J Appl Biomater Funct Mater 2014 Dec 30;12(3):155-62. Epub 2014 Dec 30.

1 Industrial Biotechnology Research Center, SIRIM Berhad, Shah Alam, Selangor - Malaysia.

Background: The physical and biological characteristics of oligochitosan (O-C) film, including its barrier and mechanical properties, in vitro cytotoxicity and in vivo biocompatibility, were studied to assess its potential use as a wound dressing.

Methods: Membrane films were prepared from water-soluble O-C solution blended with various concentrations of glycerol to modify the physical properties of the films. In vitro and in vivo biocompatibility evaluations were performed using primary human skin fibroblast cultures and subcutaneous implantation in a rat model, respectively.

Results: Addition of glycerol significantly influenced the barrier and mechanical properties of the films. Water absorption capacity was in the range of 80%-160%, whereas water vapor transmission rate varied from 1,180 to 1,618 g/m2 per day. Both properties increased with increasing glycerol concentration. Tensile strength decreased while elongation at break increased with the addition of glycerol. O-C films were found to be noncytotoxic to human fibroblast cultures and histological examination proved that films are biocompatible.

Conclusion: These results indicate that the membrane film from O-C has potential application as a wound-dressing material.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.5301/jabfm.5000190DOI Listing
December 2014

Differential protein expression between chondrogenic differentiated MSCs, undifferentiated MSCs and adult chondrocytes derived from Oryctolagus cuniculus in vitro.

Int J Med Sci 2014 11;11(1):24-33. Epub 2013 Dec 11.

1. Tissue Engineering Group (TEG), National Orthopaedic Center of Excellence for Research and Learning (NOCERAL), Department of Orthopaedic Surgery, Faculty of Medicine, University of Malaya,50603 Kuala Lumpur, Malaysia ; 3. Clinical Investigative Centre (CIC), University Malaya Medical Centre, Kuala Lumpur, Malaysia.

Objective: This preliminary study aims to determine the differentially expressed proteins from chondrogenic differentiated multipotent stromal cells (cMSCs) in comparison to undifferentiated multipotent stromal cells (MSCs) and adult chondrocytes (ACs).

Methods: ACs and bone marrow-derived MSCs were harvested from New Zealand White rabbits (n = 3). ACs and cMSCs were embedded in alginate and were cultured using a defined chondrogenic medium containing transforming growth factor-beta 3 (TGF-β3). Chondrogenic expression was determined using type-II collagen, Safranin-O staining and glycosaminoglycan analyses. Two-dimensional gel electrophoresis (2-DE) was used to isolate proteins from MSCs, cMSCs and ACs before being identified using liquid chromatography-mass spectrometry (LC-MS). The differentially expressed proteins were then analyzed using image analysis software.

Results: Both cMSCs and ACs were positively stained with type-II collagen and safranin-O. The expression of glycosaminoglycan in cMSCs was comparable to AC at which the highest level was observed at day-21 (p>0.05). Six protein spots were found to be most differentially expressed between MSCs, cMSCs and ACs. The protein spots cofilin-1 (CFL1) and glycealdehyde-3-phosphate dehydrogenase (GAPD) from cMSCs had expression levels similar to that of ACs whereas the others (ie. MYL6B, ALDOA, TAGLN2, EF1-alpha), did not match the expression level of ACs.

Conclusion: Despite having similar phenotypic expressions to ACs, cMSCs expressed proteins which were not typically expected. This may explain the reason for the unexplained lack of improvement in cartilage repair outcomes reported in previous studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7150/ijms.7244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3880988PMC
September 2014

Isolation and enhancement of a homogenous in vitro human Hertwig's epithelial root sheath cell population.

Int J Mol Sci 2013 May 27;14(6):11157-70. Epub 2013 May 27.

Conservative Department, School of Dental Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia.

Hertwig's epithelial root sheath (HERS) cells play a pivotal role during root formation of the tooth and are able to form cementum-like tissue. The aim of the present study was to establish a HERS cell line for molecular and biochemical studies using a selective digestion method. Selective digestion was performed by the application of trypsin-EDTA for 2 min, which led to the detachment of fibroblast-like-cells, with the rounded cells attached to the culture plate. The HERS cells displayed a typical cuboidal/squamous-shaped appearance. Characterization of the HERS cells using immunofluorescence staining and flow cytometry analysis showed that these cells expressed pan-cytokeratin, E-cadherin, and p63 as epithelial markers. Moreover, RT-PCR confirmed that these cells expressed epithelial-related genes, such as cytokeratin 14, E-cadherin, and ΔNp63. Additionally, HERS cells showed low expression of CD44 and CD105 with absence of CD34 and amelogenin expressions. In conclusion, HERS cells have been successfully isolated using a selective digestion method, thus enabling future studies on the roles of these cells in the formation of cementum-like tissue in vitro.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms140611157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3709725PMC
May 2013

Total cell pooling in vitro: an effective isolation method for bone marrow-derived multipotent stromal cells.

In Vitro Cell Dev Biol Anim 2013 Jun 25;49(6):424-32. Epub 2013 May 25.

Tissue Engineering Group (TEG), National Orthopaedic Centre for Research and Learning (NOCERAL), Department of Orthopaedic Surgery, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s11626-013-9626-0DOI Listing
June 2013

Supermacroporous poly(vinyl alcohol)-carboxylmethyl chitosan-poly(ethylene glycol) scaffold: an in vitro and in vivo pre-assessments for cartilage tissue engineering.

J Mater Sci Mater Med 2013 Jun 20;24(6):1561-70. Epub 2013 Mar 20.

Tissue Engineering Group-TEG, National Orthopaedic Centre for Research and Learning-NOCERAL, Department of Orthopaedic Surgery, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.

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 < 0.05). PCP scaffolds did not elicit any adverse effects on the host tissue, but were partially degraded. These results suggest that supermacroporous PCP is a biocompatible scaffold for clinical applications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10856-013-4907-4DOI Listing
June 2013

In vitro characterization of a chitosan skin regenerating template as a scaffold for cells cultivation.

Springerplus 2013 Dec 5;2(1):79. Epub 2013 Mar 5.

Reconstructive Sciences Unit, Universiti Sains Malaysia, Kelantan, Malaysia.

Chitosan is a marine-derived product that has been widely used in clinical applications, especially in skin reconstruction. The mammalian scaffolds derived from bovine and porcine material have many limitations, for example, prion transmission and religious concerns. Therefore, we created a chitosan skin regenerating template (SRT) and investigated the behavior of fibroblast cell-scaffold constructs. Primary human dermal fibroblasts (HDF) were isolated and then characterized using vimentin and versican. HDF were seeded into chitosan SRT at a density of 3×10(6) cells/cm(2) for fourteen days. Histological analysis and live cells imaging revealed that the cell-chitosan constructs within interconnected porous chitosan showed significant interaction between the cells as well as between the cells and the chitosan. Scanning electron microscopy (SEM) analysis revealed cells spreading and covering the pores. As the pore sizes of the chitosan SRT range between 40-140 μm, an average porosity is about 93 ± 12.57% and water uptake ratio of chitosan SRT is 536.02 ± 14.29%, it is a supportive template for fibroblast attachment and has potential in applications as a dermal substitute.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/2193-1801-2-79DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3597272PMC
December 2013

Keloid pathogenesis via Drosophila similar to mothers against decapentaplegic (SMAD) signaling in a primary epithelial-mesenchymal in vitro model treated with biomedical-grade chitosan porous skin regenerating template.

J Biosci Bioeng 2013 Apr 22;115(4):453-8. Epub 2012 Nov 22.

Department of Orthopaedic Surgery, University of Malaya, Kuala Lumpur, Malaysia.

The effects of locally produced chitosan (CPSRT-NC-bicarbonate) in the intervention of keloid pathogenesis were investigated in vitro. A human keratinocyte-fibroblast co-culture model was established to investigate the protein levels of human collagen type-I, III and V in a western blotting analysis, the secreted transforming growth factor-β1 (TGF-β1) in an enzyme-linked immunosorbent assay (ELISA) and the mRNA levels of TGF-β1's intracellular signaling molecules (SMAD2, 3, 4 and 7) in a real-time PCR analysis. Keratinocyte-fibroblast co-cultures were maintained in DKSFM:DMEM:F12 (2:2:1) medium. Collagen type-I was found to be the dominant form in primary normal human dermal fibroblast (pNHDF) co-cultures, whereas collagen type-III was more abundant in primary keloid-derived human dermal fibroblast (pKHDF) co-cultures. Collagen type-V was present as a minor component in the skin. TGF-β1, SMAD2 and SMAD4 were expressed more in the pKHDF than the pNHDF co-cultures. Co-cultures with normal keratinocytes suppressed collagen type-III, SMAD2, SMAD4 and TGF-β1 expressions and CPSRT-NC-bicarbonate enhanced this effect. In conclusion, the CPSRT-NC-bicarbonate in association with normal-derived keratinocytes demonstrated an ability to reduce TGF-β1, SMAD2 and SMAD4 expressions in keloid-derived fibroblast cultures, which may be useful in keloid intervention.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jbiosc.2012.10.010DOI Listing
April 2013