MyMedR

Displaying publications 21 - 40 of 81 in total

Abstract:

Sort:

New method for the isolation of endothelial cells from large vessels

Ataollahi F, Pingguan-Murphy B, Moradi A, Wan Abas WA, Chua KH, Abu Osman NA

Cytotherapy, 2014 Aug;16(8):1145-52.
PMID: 24831838 DOI: 10.1016/j.jcyt.2014.01.010

Numerous protocols for the isolation of bovine aortic endothelial cells have been described in the previous literature. However, these protocols prevent researchers from obtaining the pure population of endothelial cells. Thus, this study aimed to develop a new and economical method for the isolation of pure endothelial cells by introducing a new strategy to the enzymatic digestion method proposed by previous researchers.

Matched MeSH terms: Fibroblasts/cytology
Genotoxicity evaluation of dental restoration nanocomposite using comet assay and chromosome aberration test

Musa M, Ponnuraj KT, Mohamad D, Rahman IA

Nanotechnology, 2013 Jan 11;24(1):015105.
PMID: 23221152 DOI: 10.1088/0957-4484/24/1/015105

Nanocomposite is used as a dental filling to restore the affected tooth, especially in dental caries. The dental nanocomposite (KelFil) for tooth restoration used in this study was produced by the School of Dental Sciences, Universiti Sains Malaysia, Malaysia and is incorporated with monodispersed, spherical nanosilica fillers. The aim of the study was to determine the genotoxic effect of KelFil using in vitro genotoxicity tests. The cytotoxicity and genotoxicity of KelFil was evaluated using MTT assay, comet assay and chromosome aberration tests with or without the addition of a metabolic activation system (S9 mix), using the human lung fibroblast cell line (MRC-5). Concurrent negative and positive controls were included. In the comet assay, no comet formation was found in the KelFil groups. There was a significant difference in tail moment between KelFil groups and positive control (p < 0.05). Similarly, no significant aberrations in chromosomes were noticed in KelFil groups. The mitotic indices of treatment groups and negative control were significantly different from positive controls. Hence, it can be concluded that the locally produced dental restoration nanocomposite (KelFil) is non-genotoxic under the present test conditions.

Matched MeSH terms: Fibroblasts/cytology
Human serum is an advantageous supplement for human dermal fibroblast expansion: clinical implications for tissue engineering of skin

Mazlyzam AL, Aminuddin BS, Saim L, Ruszymah BH

Arch Med Res, 2008 Nov;39(8):743-52.
PMID: 18996287 DOI: 10.1016/j.arcmed.2008.09.001

Standard fibroblast culture medium usually contains fetal bovine serum (FBS). In theory, unknown risks of infection from bovine disease or immune reaction to foreign proteins may occur if standard culture method is used for future human tissue-engineering development. Human serum (HS) theoretically would be another choice in providing a safer approach and autologous clinically reliable cells.

Matched MeSH terms: Fibroblasts/cytology
Prospective full-term-derived pluripotent amniotic fluid stem (AFS) cells

Ferdaos N, Nathan S, Nordin N

Med J Malaysia, 2008 Jul;63 Suppl A:75-6.
PMID: 19024991

Amniotic fluid (AF) serves as an excellent alternative source of pluripotent stem cells, as they are not bound with ethical issues and the stem cells are more primitive than adult stem (AS) cells. Hence, they have higher potential. Here we aim to isolate and characterize pluripotent stem cells from mid-term and full-term pregnant rat amniotic fluid. The results demonstrate the evidence of heterogeneous population of cells in the amniotic fluid and some of the cells morphology shows similarity with ES cells.

Matched MeSH terms: Fibroblasts/cytology*
Derivation of cochlea hair cell for in vitro expansion and characterization

Ibnubaidah MA, Chua KH, Mazita A, Azida ZN, Aminuddin BS, Ruszymah BH, et al.

Med J Malaysia, 2008 Jul;63 Suppl A:115-6.
PMID: 19025012

A potential cure for hearing loss would be to regenerate hair cells by stimulating cells of the damaged inner ear sensory epithelia to proliferate and differentiate into hair cells. Here, we investigated the possibility to isolate, culture-expand and characterize the cells from the cochlea membrane of adult mice. Our results showed that the cultured cells isolated from mouse cochlea membrane were heterogenous in nature. Morphologically there were epithelial like cells, hair cell like, nerve cell like and fibroblastic cells observed in the culture. The cultured cells were immunopositive for specific hair cell markers including Myosin 7a, Calretinin and Espin.

Matched MeSH terms: Fibroblasts/cytology
Reconstruction of living bilayer human skin equivalent utilizing human fibrin as a scaffold

Mazlyzam AL, Aminuddin BS, Fuzina NH, Norhayati MM, Fauziah O, Isa MR, et al.

Burns, 2007 May;33(3):355-63.
PMID: 17321690

Our aim of this study was to develop a new methodology for constructing a bilayer human skin equivalent to create a more clinical compliance skin graft composite for the treatment of various skin defects. We utilized human plasma derived fibrin as the scaffold for the development of a living bilayer human skin equivalent: fibrin-fibroblast and fibrin-keratinocyte (B-FF/FK SE). Skin cells from six consented patients were culture-expanded to passage 1. For B-FF/FK SE formation, human fibroblasts were embedded in human fibrin matrix and subsequently another layer of human keratinocytes in human fibrin matrix was stacked on top. The B-FF/FK SE was then transplanted to athymic mice model for 4 weeks to evaluate its regeneration and clinical performance. The in vivo B-FF/FK SE has similar properties as native human skin by histological analysis and expression of basal Keratin 14 gene in the epidermal layer and Collagen type I gene in the dermal layer. Electron microscopy analysis of in vivo B-FF/FK SE showed well-formed and continuous epidermal-dermal junction. We have successfully developed a technique to engineer living bilayer human skin equivalent using human fibrin matrix. The utilization of culture-expanded human skin cells and fibrin matrix from human blood will allow a fully autologous human skin equivalent construction.

Matched MeSH terms: Fibroblasts/cytology
In vitro biocompatibility evaluation of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) copolymer in fibroblast cells

Siew EL, Rajab NF, Osman AB, Sudesh K, Inayat-Hussain SH

J Biomed Mater Res A, 2007 May;81(2):317-25.
PMID: 17120221

Among the various biomaterials available for tissue engineering and therapeutic applications, microbial polyhydroxyalkanoates offer the most diverse range of thermal and mechanical properties. In this study, the biocompatibility of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB); containing 50 mol % of 4-hydroxybutyrate] copolymer produced by Delftia acidovorans was evaluated. The cytotoxicity, mode of cell death, and genotoxicity of P(3HB-co-4HB) extract against V79 and L929 fibroblast cells were assessed using MTT assay, acridine orange/propidium iodide staining, and alkaline comet assay, respectively. Our results demonstrate that P(3HB-co-4HB) treated on both cell lines were comparable with clinically-used Polyglactin 910, where more than 60% of viable cells were observed following 72-h treatment at 200 mg/mL. Further morphological investigation on the mode of cell death showed an increase in apoptotic cells in a time-dependent manner in both cell lines. On the other hand, P(3HB-co-4HB) at 200 mg/mL showed no genotoxic effects as determined by alkaline comet assay following 72-h treatment. In conclusion, our study indicated that P(3HB-co-4HB) compounds showed good biocompatibility in fibroblast cells suggesting that it has potential to be used for future medical applications.

Matched MeSH terms: Fibroblasts/cytology
Phenotypic expression of collagen type II and collagen type I gene in monolayer culture of human auricular chondrocytes

Nur Adelina AN, Aminuddin BS, Munirah S, Chua KH, Fuzina NH, Saim L, et al.

Med J Malaysia, 2004 May;59 Suppl B:188-9.
PMID: 15468881

Cartilage is regularly needed for reconstructive surgery. Basic research in tissue engineering is necessary to develop its full potential. We presented here the expression profile of type II collagen gene and type I collagen gene in human auricular monolayer culture expansion. Cultured chondrocytes documented a reduction in the expression level of collagen type II gene whilst collagen type I gene was gradually expressed through all the passages. This study demonstrated that human auricular chondrocytes lose its phenotypic expression during monolayer culture expansion. Further studies are required to enhance cartilage specific gene expression, collagen type II throughout the in vitro culture.

Matched MeSH terms: Fibroblasts/cytology
Comparison of chitosan scaffold and chitosan-collagen scaffold: a preliminary study

Norazril SA, Aminuddin BS, Norhayati MM, Mazlyzam AL, Fauziah O, Ruszymah BH

Med J Malaysia, 2004 May;59 Suppl B:186-7.
PMID: 15468880

Chitosan has similar structure to glycosaminoglycans in the tissue, thus may be a good candidates as tissue engineering scaffold. However, to improve their cell attachment ability, we try to incorporate this natural polymer with collagen by combining it via cross-linking process. In this preliminary study we evaluate the cell attachment ability of chitosan-collagen scaffold versus chitosan scaffold alone. Chitosan and collagen were dissolved in 1% acetic acid and then were frozen for 24 hours before the lyophilizing process. Human skin fibroblasts were seeded into both scaffold and were cultured in F12: DMEM (1:1). Metabolic activity assay were used to evaluate cell attachment ability of scaffold for a period of 1, 3, 7 and 14 days. Scanning electron micrographs shows good cell morphology on chitosan-collagen hybrid scaffold. In conclusion, the incorporation of collagen to chitosan will enhance its cell attachment ability and will be a potential scaffold in tissue engineering.

Matched MeSH terms: Fibroblasts/cytology
Tissue engineered trachea using sheep nasal septum

Kojima K

Med J Malaysia, 2004 May;59 Suppl B:32-3.
PMID: 15468805
Matched MeSH terms: Fibroblasts/cytology
Fulltext In Vitro Study of Surface Modified Poly(ethylene glycol)-Impregnated Sintered Bovine Bone Scaffolds on Human Fibroblast Cells

Pramanik S, Ataollahi F, Pingguan-Murphy B, Oshkour AA, Osman NAA

Sci Rep, 2015 May 07;5:9806.
PMID: 25950377 DOI: 10.1038/srep09806

Scaffold design from xenogeneic bone has the potential for tissue engineering (TE). However, major difficulties impede this potential, such as the wide range of properties in natural bone. In this study, sintered cortical bones from different parts of a bovine-femur impregnated with biodegradable poly(ethylene glycol) (PEG) binder by liquid phase adsorption were investigated. Flexural mechanical properties of the PEG-treated scaffolds showed that the scaffold is stiffer and stronger at a sintering condition of 1000°C compared with 900°C. In vitro cytotoxicity of the scaffolds evaluated by Alamar Blue assay and microscopic tests on human fibroblast cells is better at 1000°C compared with that at 900°C. Furthermore, in vitro biocompatibility and flexural property of scaffolds derived from different parts of a femur depend on morphology and heat-treatment condition. Therefore, the fabricated scaffolds from the distal and proximal parts at 1000°C are potential candidates for hard and soft TE applications, respectively.

Matched MeSH terms: Fibroblasts/cytology*
Tocotrienol-rich fraction prevents cellular aging by modulating cell proliferation signaling pathways

Khor SC, Mohd Yusof YA, Wan Ngah WZ, Makpol S

Clin Ter, 2015;166(2):e81-90.
PMID: 25945449 DOI: 10.7417/CT.2015.1825

BACKGROUND AND OBJECTIVE: Vitamin E has been suggested as nutritional intervention for the prevention of degenerative and age-related diseases. In this study, we aimed to elucidate the underlying mechanism of tocotrienol-rich fraction (TRF) in delaying cellular aging by targeting the proliferation signaling pathways in human diploid fibroblasts (HDFs).
MATERIALS AND METHODS: Tocotrienol-rich fraction was used to treat different stages of cellular aging of primary human diploid fibroblasts viz. young (passage 6), pre-senescent (passage 15) and senescent (passage 30). Several selected targets involved in the downstream of PI3K/AKT and RAF/MEK/ERK pathways were compared in total RNA and protein.
RESULTS: Different transcriptional profiles were observed in young, pre-senescent and senescent HDFs, in which cellular aging increased AKT, FOXO3, CDKN1A and RSK1 mRNA expression level, but decreased ELK1, FOS and SIRT1 mRNA expression level. With tocotrienol-rich fraction treatment, gene expression of AKT, FOXO3, ERK and RSK1 mRNA was decreased in senescent cells, but not in young cells. The three down-regulated mRNA in cellular aging, ELK1, FOS and SIRT1, were increased with tocotrienol-rich fraction treatment. Expression of FOXO3 and P21Cip1 proteins showed up-regulation in senescent cells but tocotrienol-rich fraction only decreased P21Cip1 protein expression in senescent cells.
CONCLUSIONS: Tocotrienol-rich fraction exerts gene modulating properties that might be responsible in promoting cell cycle progression during cellular aging.

Matched MeSH terms: Fibroblasts/cytology
Fulltext Chitosan dermal substitute and chitosan skin substitute contribute to accelerated full-thickness wound healing in irradiated rats

Mohd Hilmi AB, Halim AS, Jaafar H, Asiah AB, Hassan A

Biomed Res Int, 2013;2013:795458.
PMID: 24324974 DOI: 10.1155/2013/795458

Wounds with full-thickness skin loss are commonly managed by skin grafting. In the absence of a graft, reepithelialization is imperfect and leads to increased scar formation. Biomaterials can alter wound healing so that it produces more regenerative tissue and fewer scars. This current study use the new chitosan based biomaterial in full-thickness wound with impaired healing on rat model. Wounds were evaluated after being treated with a chitosan dermal substitute, a chitosan skin substitute, or duoderm CGF. Wounds treated with the chitosan skin substitute showed the most re-epithelialization (33.2 ± 2.8%), longest epithelial tongue (1.62 ± 0.13 mm), and shortest migratory tongue distance (7.11 ± 0.25 mm). The scar size of wounds treated with the chitosan dermal substitute (0.13 ± 0.02 cm) and chitosan skin substitute (0.16 ± 0.05 cm) were significantly decreased (P < 0.05) compared with duoderm (0.45 ± 0.11 cm). Human leukocyte antigen (HLA) expression on days 7, 14, and 21 revealed the presence of human hair follicle stem cells and fibroblasts that were incorporated into and surviving in the irradiated wound. We have proven that a chitosan dermal substitute and chitosan skin substitute are suitable for wound healing in full-thickness wounds that are impaired due to radiation.

Matched MeSH terms: Fibroblasts/cytology
A two-step method using air plasma and carbodiimide crosslinking to enhance the biocompatibility of polycaprolactone

Sahapaibounkit P, Prasertsung I, Mongkolnavin R, Wong CS, Damrongsakkul S

J Biomed Mater Res B Appl Biomater, 2017 08;105(6):1658-1666.
PMID: 27177842 DOI: 10.1002/jbm.b.33708

In this study, polycaprolactone (PCL) film, a high potential material used in biomedical applications, was treated by air plasma prior to a conjugation by carbodiimide cross-linking with various types of proteins, including type A gelatin, type B gelatin, and collagen hydrolysate. The properties of modified PCL films were characterized by X-ray photoelectron spectroscopy (XPS), contact angle measurement, and atomic force microscopy. The XPS results showed that oxygen and nitrogen atoms were successfully introduced on the air plasma-treated PCL surface. Primary amine was found on the air plasma-treated PCL films. All proteins were shown to be successfully cross-linked on air plasma-treated PCL films. The wettability and roughness of protein-conjugated PCL films were significantly increased compared to those of neat PCL film. In vitro biocompatibility test using L929 mouse fibroblast showed that the attachment percentage and spreading area of attached cells on all protein-conjugated PCL films were markedly increased. Comparing among modified PCL films, no significant difference on the attachment of L929 on modified PCL films was noticed. However, the spreading areas of cells after 24 hours of culture on type A gelatin- and type B gelatin-modified PCL surfaces were higher than that on collagen hydrolysate-modified surface, possibly related to the lower percentage of amide bond on collagen hydrolysate-conjugated surface compared to those on both gelatin-conjugated PCL ones. This indicated that the two-step modification of PCL film via air plasma and carbodiimide cross-linking with collagen-derived proteins could enhance the biocompatibility of PCL films. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1658-1666, 2017.

Matched MeSH terms: Fibroblasts/cytology
Comparative efficacy of hemorrhage control of a novel mesoporous bioactive glass versus two commercial hemostats

Pourshahrestani S, Kadri NA, Zeimaran E, Gargiulo N, Samuel S, Naveen SV, et al.

Biomed Mater, 2018 02 08;13(2):025020.
PMID: 29148431 DOI: 10.1088/1748-605X/aa9b3e

Mesoporous bioactive glass containing 1% Ga2O3 (1%Ga-MBG) is attractive for hemorrhage control because of its surface chemistry which can promote blood-clotting. The present study compares this proprietary inorganic coagulation accelerator with two commercial hemostats, CeloxTM (CX) and QuikClot Advanced Clotting Sponge PlusTM (ACS+). The results indicate that the number of adherent platelets were higher on the 1%Ga-MBG and CX surfaces than ACS+ whereas a greater contact activation was seen on 1%Ga-MBG and ACS+ surfaces than CX. 1%Ga-MBG not only resulted in larger platelet aggregates and more extensive platelet pseudopodia compared to CX and ACS+ but also significantly accelerated the intrinsic pathways of the clotting cascade. In vitro thrombin generation assays also showed that CX and ACS+ induced low levels of thrombin formation while 1%Ga-MBG had significantly higher values. 1%Ga-MBG formed a larger red blood cell aggregate than both CX and ACS+. Direct exposure of 1%Ga-MBG to fibroblast cells increased cell viability after 3 days relative to CX and ACS+, inferring excellent cytocompatibility. The results of this study promote 1%Ga-MBG as a promising hemostat compared to the commercially available products as it possesses essential factors required for coagulation activation.

Matched MeSH terms: Fibroblasts/cytology
Decellularization and genipin crosslinking of amniotic membrane suitable for tissue engineering applications

Gobinathan S, Zainol SS, Azizi SF, Iman NM, Muniandy R, Hasmad HN, et al.

J Biomater Sci Polym Ed, 2018 12;29(17):2051-2067.
PMID: 29983100 DOI: 10.1080/09205063.2018.1485814

Amniotic membrane has the potential to be used as scaffold in various tissue engineering applications. However, increasing its biostability at the same time maintaining its biocompatibility is important to enhance its usage as a scaffold. This studied characteristics genipin-crosslinked amniotic membrane as a bioscaffold. Redundant human amniotic membranes (HAM) divided into native (nAM), decellularized (dAM) and genipin-crosslinked (clAM) groups. The dAM and clAM group were decellularized using thermolysin (TL) and sodium hydroxide (NaOH) solution. Next, clAM group was crosslinked with 0.5% and 1.0% (w/v) genipin. The HAM was then studied for in vitro degradation, percentage of swelling, optical clarity, ultrastructure and mechanical strength. Meanwhile, fibroblasts isolated from nasal turbinates were then seeded onto nAM, dAM and clAM for biocompatibility studies. clAM had the slowest degradation rate and were still morphologically intact after 30 days of incubation in 0.01% collagenase type 1 solution. The dAM had a significantly highest percentage of swelling than other groups (p

Matched MeSH terms: Fibroblasts/cytology
Fulltext Nanolayered composite with enhanced ultraviolet ray absorption properties from simultaneous intercalation of sunscreen molecules

Megat Nabil Mohsin S, Hussein MZ, Sarijo SH, Fakurazi S, Arulselvan P, Taufiq-Yap YH

Int J Nanomedicine, 2018;13:6359-6374.
PMID: 30349255 DOI: 10.2147/IJN.S171390

Introduction: The potential of layered double hydroxide (LDH) as a host of multiple ultraviolet-ray absorbers was investigated by simultaneous intercalation of benzophenone 4 (B4) and Eusolex® 232 (EUS) in Zn/Al LDH.
Methods: The nanocomposites were prepared via coprecipitation method at various molar ratios of B4 and EUS.
Results: At equal molar ratios, the obtained nanocomposite showed an intercalation selectivity that is preferential to EUS. However, the selectivity ratio of intercalated anions was shown to be capable of being altered by adjusting the molar ratio of intended guests during synthesis. Dual-guest nanocomposite synthesized with B4:EUS molar ratio 3:1 (ZEB [3:1]) showed an intercalation selectivity ratio of B4:EUS =53:47. Properties of ZEB (3:1) were monitored using powder X-ray diffractometer to show a basal spacing of 21.8 Å. Direct-injection mass spectra, Fourier transform infrared spectra, and ultraviolet-visible spectra confirmed the dual intercalation of both anions into the interlayer regions of dual-guest nanocomposite. The cytotoxicity study of dual-guest nanocomposite ZEB (3:1) on human dermal fibroblast cells showed no significant toxicity until 25 μg/mL.
Conclusion: Overall, the findings demonstrate successful customization of ultraviolet-ray absorbers composition in LDH host.

Matched MeSH terms: Fibroblasts/cytology
Fulltext Proteomic profiling of senescent human diploid fibroblasts treated with gamma-tocotrienol

Tan JK, Jaafar F, Makpol S

BMC Complement Altern Med, 2018 Nov 29;18(1):314.
PMID: 30497457 DOI: 10.1186/s12906-018-2383-6

BACKGROUND: Replicative senescence of human diploid fibroblasts (HDFs) has been used as a model to study mechanisms of cellular aging. Gamma-tocotrienol (γT3) is one of the members of vitamin E family which has been shown to increase proliferation of senescent HDFs. However, the modulation of protein expressions by γT3 in senescent HDFs remains to be elucidated. Therefore, this study aimed to determine the differentially expressed proteins (DEPs) in young and senescent HDFs; and in vehicle- and γT3-treated senescent HDFs using label-free quantitative proteomics.
METHODS: Whole proteins were extracted and digested in-gel with trypsin. Peptides were detected by Orbitrap liquid chromatography mass spectrometry. Mass spectra were identified and quantitated by MaxQuant software. The data were further filtered and analyzed statistically using Perseus software to identify DEPs. Functional annotations of DEPs were performed using Panther Classification System.
RESULTS: A total of 1217 proteins were identified in young and senescent cells, while 1218 proteins in vehicle- and γT3-treated senescent cells. 11 DEPs were found in young and senescent cells which included downregulation of platelet-derived growth factor (PDGF) receptor beta and upregulation of tubulin beta-2A chain protein expressions in senescent cells. 51 DEPs were identified in vehicle- and γT3-treated senescent cells which included upregulation of 70 kDa heat shock protein, triosephosphate isomerase and malate dehydrogenase protein expressions in γT3-treated senescent cells.
CONCLUSIONS: PDGF signaling and cytoskeletal structure may be dysregulated in senescent HDFs. The pro-proliferative effect of γT3 on senescent HDFs may be mediated through the stimulation of cellular response to stress and carbohydrate metabolism. The expressions and roles of these proteins in relation to cellular senescence are worth further investigations. Data are available via ProteomeXchange with identifier PXD009933.

Matched MeSH terms: Fibroblasts/cytology
Fulltext Development of a luciferase/luciferin cell proliferation (XenoLuc) assay for real-time measurements of Gfp-Luc2-modified cells in a co-culture system

Teow SY, Liew K, Che Mat MF, Marzuki M, Abdul Aziz N, Chu TL, et al.

BMC Biotechnol, 2019 06 14;19(1):34.
PMID: 31200673 DOI: 10.1186/s12896-019-0528-4

BACKGROUND: In vitro modelling of cancer cells is becoming more complex due to prevailing evidence of intimate interactions between cancer cells and their surrounding stroma. A co-culture system which consists of more than one cell type is physiologically more relevant and thus, could serve as a useful model for various biological studies. An assay that specifically detects the phenotypic changes of cancer cells in a multi-cellular system is lacking for nasopharyngeal carcinoma (NPC).
RESULTS: Here, we describe a luciferase/luciferin (XenoLuc) assay that could specifically measure changes in the proliferation of cancer cells in the co-culture system using two modified NPC patient-derived tumour xenograft (PDTXs) cells: Xeno284-gfp-luc2 and XenoB110-gfp-luc2. Through this assay, we are able to show that the growth of NPC xenograft cells in both two-dimensional (2D) and three-dimensional (3D) models was enhanced when co-cultured with normal human dermal fibroblasts (NHDFs). In addition, potential applications of this assay in in vitro drug or inhibitor screening experiments are also illustrated.
CONCLUSIONS: XenoLuc assay is specific, sensitive, rapid and cost-effective for measuring the growth of luciferase-expressing cells in a co- or multiple-culture system. This assay may also be adapted for tumour microenvironment studies as well as drug screening experiments in more complex 3D co-culture systems.

Matched MeSH terms: Fibroblasts/cytology
Matrix Production in Chondrocytes Transfected with Sex Determining Region Y-Box 9 and Telomerase Reverse Transcriptase Genes: An In Vitro Evaluation from Monolayer Culture to Three-Dimensional Culture

Md Nazir N, Zulkifly AH, Khalid KA, Zainol I, Zamli Z, Sha'ban M

Tissue Eng Regen Med, 2019 06;16(3):285-299.
PMID: 31205857 DOI: 10.1007/s13770-019-00191-1

Background: This study aimed to observe the cartilaginous matrix production in SRY (sex determining region Y)-box 9 (SOX9)- and/or telomerase reverse transcriptase (TERT)-transfected chondrocytes from monolayer to three-dimensional (3D) culture.
Methods: The genes were transferred into chondrocytes at passage-1 (P1) via lipofection. The post-transfected chondrocytes (SOX9-, TERT- and SOX9/TERT) were analysed at P1, P2 and P3. The non-transfected group was used as control. The 3D culture was established using the chondrocytes seeded in a disc-shaped PLGA/fibrin and PLGA scaffolds. The resulting 3D "cells-scaffolds" constructs were analysed at week-1, -2 and -3. The histoarchitecture was evaluated using haematoxylin and eosin, alcian blue and safranin o stains. The quantitative sulphated glycosaminoglycan (sGAG) content was measured using biochemical assay. The cartilage-specific markers expression were analysed via real-time polymerase chain reaction.
Results: All monolayer cultured chondrocytes showed flattened, fibroblast-like appearance throughout passages. Proteoglycan and sGAG were not detected at the pericellular matrix region of the chondrocytes. The sGAG content assay indicated the matrix production depletion in the culture. The cartilage-specific markers, COL2A1 and ACAN, were downregulated. However, the dedifferentiation marker, COL1A1 was upregulated. In 3D "cells-scaffolds" constructs, regardless of transfection groups, chondrocytes seeded in PLGA/fibrin showed a more uniform distribution and produced denser matrix than the PLGA group especially at week-3. Both sGAG and proteoglycan were clearly visualised in the constructs, supported by the increment of sGAG content, quantitatively. Both COL2A1 and ACAN were upregulated in SOX9/TERT-PLGA and SOX9/TERT-PLGA/fibrin respectively. While, COL1A1 was downregulated in SOX9/TERT-PLGA.
Conclusion: These findings indicated that the SOX9/TERT-transfected chondrocytes incorporation into 3D scaffolds facilitates the cartilage regeneration which is viable structurally and functionally.

Matched MeSH terms: Fibroblasts/cytology

Filters

Please provide feedback to Administrator (afdal@afpm.org.my)

External Links