[Show abstract][Hide abstract] ABSTRACT: Graphene can induce osteogenic differentiation of stem cells. However, the cellular mechanisms involved in this process remain unexplored. Our objective was to investigate key factors, in both genomic and protein level, involved in the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) in two and three-dimensional graphene substrates. PDLSC were seeded on glass slides (Gl); Gl coated with graphene (2DGp), three-dimensional graphene scaffold (3DGp) and polystyrene scaffold (PS) and cultured with and without osteogenic medium for 28 days. All the substrates allowed stem cell survival and proliferation. 2DGp and 3DGp induced the differentiation of PDLSC into mature osteoblasts at higher levels as compared to Gl and PS. Bone-related gene and proteins (COL I, RUNX2, OCN) were upregulated on graphene regardless the use of osteogenic medium. The high expression of MHY10 and MHY10-V2 on 2DGp and 3DGp suggest that their physical characteristics may play a role in the enhanced differentiation. As the results were boosted by the use of osteogenic medium, we suggest that both chemical and physical properties of graphene act synergistically while ruling osteoblastic differentiation of PDLSC.
[Show abstract][Hide abstract] ABSTRACT: Objective To investigate the physical (setting time, hardness, flowability, microstructure) and chemical (pH change, calcium release, crystallinity) properties and the biological outcomes (cell survival and differentiation) of mineral trioxide aggregate (MTA) mixed using different proportions of propylene glycol (PG) and water.Material and Methods White MTA was mixed with different water/PG ratios (100/0, 80/20 and 50/50). Composition (XRD), microstructure (SEM), setting time (ASTM C266-13), flowability (ANSI/ADA 57-2000), Knoop hardness (100 g/10 s) and chemical characteristics (pH change and Ca2+ release for 7 days) were evaluated. Cell proliferation, osteo/odontoblastic gene expression and mineralization induced by MTA mixed with PG were evaluated. MTA discs (5 mm in diameter, 2 mm thick) were prepared and soaked in culture medium for 7 days. Next, the discs were removed and the medium used to culture dental pulp stem cells (DPSC) for 28 days. Cells survival was evaluated using MTS assay (24, 72 and 120 h) and differentiation with RT-PCR (ALP, OCN, Runx2, DSPP and MEPE) and alizarin red staining (7 and 14 days). Data were analysed using one-way ANOVA and Tukey's post-hoc analysis (a=0.05).Results The addition of PG significantly increased setting time, flowability and Ca2+ release, but it compromised the hardness of the material. SEM showed that 50/50 group resulted porous material after setting due to the incomplete setting reaction, as shown by XRD analysis. The addition of PG (80/20 and 50/50) was not capable to improve cell proliferation or to enhance gene expression, and mineralized deposition of DPSC after 7 and 14 days as compared to the 100/0.Conclusion Except for flowability, the addition of PG did not promote further improvements on the chemical and physical properties evaluated, and it was not capable of enhancing the bioactivity of the MTA.
Journal of applied oral science: revista FOB 09/2015; 23(4):405-411. DOI:10.1590/1678-775720150084 · 0.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Previously, we demonstrated that undifferentiated human embryonic stem cells (hESC) displayed higher resistance to oxidative and genotoxic stress compared to somatic cells, but did not further probe the underlying mechanisms. Using H₂O₂-induced genotoxicity as a model, this study investigated whether higher resistance of hESC to oxidative and genotoxic stress could be due to lower innate basal intracellular levels of reactive oxygen species (ROS), as compared to their differentiated fibroblastic progenies (H1F) and two other somatic cell types - human embryonic palatal mesenchymal (HEPM) cells and peripheral blood lymphocytes (PBL). Comet assay demonstrated that undifferentiated hESC consistently sustained lower levels of DNA damage upon acute exposure to H₂O₂ for 30 min, compared to somatic cells. DCFDA and HE staining with flow cytometry showed that undifferentiated hESC had lower innate basal intracellular levels of reactive oxygen species compared to somatic cells, which could lead to their higher resistance to genotoxic stress upon acute exposure to H₂O₂.
[Show abstract][Hide abstract] ABSTRACT: The development of materials and strategies that can influence stem cell attachment, proliferation, and differentiation towards osteoblasts is of high interest to promote faster healing and reconstructions of large bone defects. Graphene and its derivatives (graphene oxide and reduced graphene oxide) have received increasing attention for biomedical applications as they present remarkable properties such as high surface area, high mechanical strength, and ease of functionalization. These biocompatible carbon-based materials can induce and sustain stem cell growth and differentiation into various lineages. Furthermore, graphene has the ability to promote and enhance osteogenic differentiation making it an interesting material for bone regeneration research. This paper will review the important advances in the ability of graphene and its related forms to induce stem cells differentiation into osteogenic lineages.
Stem cell International 06/2015; 2015. DOI:10.1155/2015/804213 · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Culture microenvironment plays a critical role in the propagation and differentiation of human embryonic stem cells (hESCs) and their differentiated progenies. Although high efficiency of hESC differentiation to keratinocytes (hESC-Kert) has been achieved, little is known regarding the effects of early culture microenvironment and pertinent extracellular matrix (ECM) interactions during epidermal commitment on subsequent proliferative capacity of hESC-Kert. The aim of this study is to evaluate the effects of the different ECM microenvironments during hESC differentiation on subsequent replicative life span of hESC-Kert. In doing so, H1-hESCs were differentiated to keratinocytes (H1-Kert) in two differentiation systems. The first system employed autologous fibroblast feeder support, in which keratinocytes (H1-KertACC) were derived by coculture of hESCs with hESC-derived fibroblasts (H1-ebFs). The second system employed a novel decellularized matrix from H1-ebFs to create a dermoepidermal junction-like (DEJ) matrix. H1-KertAFF were derived by differentiation of hESCs on the feeder-free system employing the DEJ matrix. Our study indicated that the feeder-free system with the use of DEJ matrix was more efficient in differentiation of hESCs toward epidermal progenitors. However, the feeder-free system was not sufficient to support the subsequent replicative capacity of differentiated keratinocytes. Of note, H1-KertAFF showed limited replicative capacity with reduced telomere length and early cellular senescence. We further showed that the lack of cell-cell interactions during epidermal commitment led to heightened production of TGF-β1 by hESC-Kert during extended culture, which in turn was responsible for resulting in the limited replicative life span with cellular senescence of hESC-Kert derived under the feeder-free culture system. This study highlights for the first time the importance of the culture microenvironment and cell-ECM interactions during differentiation of hESCs on subsequent replicative life span and cellular senescence of the differentiated keratinocytes, with implications for use of these cells for applications in tissue engineering and regenerative medicine.
Tissue Engineering Part A 02/2015; 21(7-8). DOI:10.1089/ten.TEA.2014.0551 · 4.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The epidermis is mainly composed of keratinocytes forming a protective barrier. It is perpetually subjected to mechanical stress and strain during development, homeostasis and disease. Perturbation of the normal strain with alteration of its biological response may lead to severe diseases such as psoriasis and epidermolysis bullosa. To date, most of the studies about skin response to mechanical stress used immor-talized cell lines (i.e. HaCaT) or primary cells from donors, which suffer issues of limited physiological relevance and inter-donor variability. It is therefore necessary to develop a new human model for the study of normal skin physiology and response to mechanical stress. In this study, we investi-gated the use of keratinocytes derived from human embryonic stem cells (hESCs) as a reliable alternative model to HaCaT for study of the effects of mechanical tension. With compar-ison to HaCaT, hESC-derived keratinocytes (hESC-Kert) were exposed to up to 3 days of cyclic mechanical stress, and gene expression changes were analyzed. Dynamic expres-sion of several key mechanical stress related-genes was stud-ied at mRNA level using qPCR. The expression of matrix-metallopeptidase9 was studied at protein level using ELISA. The two cell types displayed similar gene expression kinetics for most of the genes including E-cadherin, cateninβ1, connexin43, desmoglein1, endothelin1, integrinα6, interleukinα1, keratin1, 6, and 10, keratinocyte growth-factor-receptor and lamininα5. Unlike HaCaT, hESC-Kert displayed early gene and protein expression of matrix metallopeptidase 9 following mechanical stimulation, sug-gesting that these cells have remodeling capacity that resem-bles that of normal human skin. Our study confirmed the use of hESC-Kert as a good model for study of skin response to mechanical stress.
[Show abstract][Hide abstract] ABSTRACT: To engineer constructs of the periodontal ligament (PDL), human PDL cells were incorporated into a matrix of hyaluronan, gelatin and type I collagen in sample holders (13 mm x 1mm) of 6-well Biopress culture plates. The loading dynamics of the PDL were mimicked by applying a cyclic compressive strain of 33.4 kPa (340.6 gm/cm2) to the constructs for 1.0 sec every 60 sec, for 6, 12 and 24h in a Flexercell FX-4000C Strain Unit. Compression significantly increased the number of nonviable cells and increased the expression of several apoptosis-related genes, including initiator and executioner caspases. Of the 15 extracellular matrix genes screened, most were upregulated at some point after 6‒12h deformation, but all were down-regulated at 24h except for MMPs1‒3 and CTGF. In culture supernatants, MMP-1 and TIMP-1 protein levels were upregulated at 24h; RANKL, OPG and FGF-2 were unchanged; and CTGF not detected. The low modulus of elasticity of the constructs was a disadvantage ‒ future mechanobiology studies and tissue engineering applications will require constructs with much higher stiffness. Since the major structural protein of the PDL is type I collagen, a more rational approach would be to permeabilize preformed type I collagen scaffolds with PDL-populated matricies.
Tissue Engineering Part A 09/2014; 21(3-4). DOI:10.1089/ten.TEA.2014.0221 · 4.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human embryonic stem cells (hESCs) have the ability to self-renew and differentiate into any cell lineage of the three germ layers, therefore holding great promise for regenerative applications in dentistry and medicine. We previously described a micromass culture system as a model system to induce and study the chondrogenic commitment of hESCs. Using this system, chondrogenic cells can be further isolated and expanded under specific growth factor conditions. When encapsulated in hyaluronic acid (HA)-based hydrogels and cultured under appropriate growth factor and medium conditions, these chondrogenic cells synthesized and deposited extracellular matrix (ECM) characteristic of neocartilage. Here, we describe the micromass culture of hESCs, the isolation and expansion of hESC-derived chondrogenic cells, and the three-dimensional (3-D) culture of the chondrogenic cells in hydrogels for cartilage tissue engineering. We will also describe the various tools and techniques used for characterizing the tissue-engineered cartilage.
[Show abstract][Hide abstract] ABSTRACT: Objective:
The role of subcutaneous adipose tissue (SAT) in the pathogenesis of type 2 diabetes is still under controversy. In this study, the metabolic effects of inducing adipocyte hyperplasia in SAT depots in obese mice were investigated.
High fat diet was used to induce obesity and type 2 diabetes symptoms in C57BL6/J mice. To induce SAT expansion through hyperplasia, acellular adipogenic cocktails were injected around the SAT depots in high fat diet-induced obese mice.
Ten weeks after injections, significant neoadipogenesis was induced, which not only obviously expanded the volume of SATs but also significantly increased the adipocyte density within the whole SAT depots. Importantly, these mice exhibited improved glucose tolerance and insulin sensitivity (homeostatic model assessment) when compared to control group. Further studies suggested that these beneficial metabolic effects were associated with elevation of serum high-molecular-weight adiponectin level and reduction of ectopic lipid accumulation in liver.
These findings not only further supported the protective role of SAT in the pathogenesis of type 2 diabetes but also highlighted the importance of adipocyte hyperplasia in this protective effect.
[Show abstract][Hide abstract] ABSTRACT: This study evaluated human embryonic stem cells (hESC) and their differentiated fibroblastic progenies as cellular models for genotoxicity screening. The DNA damage response of hESCs and their differentiated fibroblastic progenies were compared to a fibroblastic cell line (HEPM, CRL1486) and primary cultures of peripheral blood lymphocytes (PBL), upon exposure to Mitomycin C, gamma irradiation and H2O2. It was demonstrated that hESC-derived fibroblastic progenies (H1F) displayed significantly higher chromosomal aberrations, micronuclei formation and double strand break (DSB) formation, as compared to undifferentiated hESC upon exposure to genotoxic stress. Nevertheless, H1F cell types displayed comparable sensitivities to genotoxic challenge as HEPM and PBL, both of which are representative of somatic cell types commonly used for genotoxicity screening. Subsequently, transcriptomic and pathways analysis identified differential expression of critical genes involved in cell death and DNA damage response upon exposure to gamma irradiation. The results thus demonstrate that hESC-derived fibroblastic progenies are as sensitive as commonly-used somatic cell types for genotoxicity screening. Moreover, hESCs have additional advantages, such as their genetic normality compared to immortalized cell lines, as well as their amenability to scale-up for producing large, standardized quantities of cells for genotoxicity screening on an industrial scale, something which can never be achieved with primary cell cultures.
Journal of Biotechnology 05/2014; DOI:10.1016/j.jbiotec.2014.05.009 · 2.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Introduction:
The induced pluripotent stem cells (iPSCs) have characteristics similar to embryonic stem cells, including the capability of self-renewal and large-scale expansion and the ability to differentiate into all types of cells including germ cells, which defines pluripotency. Using iPSC avoids problems of immunological rejection and ethical controversy. The possible future uses of iPSC are diverse and go beyond the differentiation into somatic cells for regeneration of damaged tissues.
A unique feature of iPSC is the potential to generate patient disease-specific tissues. Thus, cells from patients can be differentiated into relevant cells of interest for drug screening, characterization of drug effects and cytotoxic assays. This review presents key aspects related to iPSC, such as their generation, potential for disease modeling, treatment, drug development and future contributions to the craniofacial complex.
It is undisputable that the evolution in iPSC knowledge will improve the approaches for drug screening and development, help to understand and treat disease origins and mechanisms and provide new strategies to clinical treatment. However, it is necessary to fine-tune protocols to establish iPSCs that are cost-effective and safe for clinical use.
[Show abstract][Hide abstract] ABSTRACT: For many years, cell therapies have been hampered by limited availability and inter-batch variability of primary cells. Human embryonic stem cell (hESC) can give rise to specialized cells like keratinocytes and recently emerged as a virtually unlimited source of potential therapeutic cells. However, xenogeneic components in differentiation cocktails have been limiting the clinical potential of hESC-derived keratinocytes (hESCs-Kert). Here, we demonstrated efficient differentiation of H9 human embryonic stem cells (H9-hESCs) into keratinocytes (H9-KertACC) in an autogenic co-culture system. We used activin as the main factor to induce keratinocyte differentiation. H9-KertACC expressed keratinocyte markers at mRNA and protein levels. Establishment of such animal-free microenvironment for keratinocyte differentiation will accelerate potential clinical application of hESCs.
Methods in molecular biology (Clifton, N.J.), 11/2013: chapter Differentiation of Epidermal Keratinocytes from Human Embryonic Stem Cells; Springer.
[Show abstract][Hide abstract] ABSTRACT: Scaffolds based on decellularized adipose tissue (DAT) are gaining popularity in adipose tissue engineering due to their high biocompatibility and adipogenic inductive property. However, previous studies involving DAT-derived scaffolds have not fully revealed their potentials for in vivo adipose tissue construction. With the aim of developing a more efficient adipose tissue engineering technique based on DAT, in this study, we investigated the in vivo adipogenic potential of a basic fibroblast growth factor (bFGF) delivery system based on heparinized DAT (Hep-DAT). To generate this system, heparins were cross-linked to mouse DATs by using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide and N-Hydroxysuccinimide. The bFGF-binding Hep-DATs were first tested for controlled release ability in vitro and then transplanted subcutaneously. Highly vascularized adipose tissues were formed 6weeks after transplantation. Histology and gene expression analysis revealed that majority of the Hep-DAT scaffolds were infiltrated with host-derived adipose tissues that possessed similar adipogenic and inflammatory gene expression as endogenous adipose tissues. Additionally, strong de novo adipogenesis could also be induced when bFGF-binding Hep-DATs were thoroughly minced and injected subcutaneously. In conclusion, our study demonstrated that bFGF-binding Hep-DAT could be an efficient, biocompatible and injectable adipogenic system for in vivo adipose tissue engineering.
[Show abstract][Hide abstract] ABSTRACT: To understand, in greater detail, the molecular mechanisms regulating the complex relationship between mechanical strain and alveolar bone metabolism during orthodontic treatment, passive cross-arch palatal springs were bonded to the maxillary molars of 6-wk-old rats, which were killed after 4 and 8 d. Outcome measures included serum assays for markers of bone formation and resorption and for the multifunctional hormone leptin, and histomorphometry of the inter-radicular bone. The concentration of the bone-formation marker alkaline phosphatase (ALP) was significantly reduced at both time points in the appliance group, accompanied by a 50% reduction in inter-radicular bone volume; however, osteocalcin (bone Gla protein) levels remained unaffected. Bone collagen deoxypyridinoline (DPD) crosslinks increased 2.3-fold at 4 d only, indicating a transient increase in bone resorption; in contrast, the level of the osteoclast-specific marker, tartrate-resistant acid phosphatase 5b (TRACP 5b), was unchanged. Leptin levels closely paralleled ALP reductions at both time points, suggesting an important role in the mechanostat negative-feedback loop required to normalize bone mass. These data suggest that an orthodontic appliance, in addition to remodeling the periodontal ligament (PDL)-bone interface, may exert unexpected side-effects on the tooth-supporting alveolar bone, and highlights the importance of recognizing that bone strains can have negative, as well as positive, effects on bone mass.
European Journal Of Oral Sciences 09/2013; 121(6). DOI:10.1111/eos.12091 · 1.49 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to differentiate human embryonic stem cells (hESCs) into odontoblastic lineage in an optimized culture milieu.
In Phase 1, hESCs were differentiated into mesenchymal stem cells (H9-MSCs). In Phase 2, H9-MSCs were then differentiated into odontoblast-like cells (H9-Odont) under the stimulation of FGF-8 and BMP-4. Alternatively, H9-MSCs were differentiated into osteogenic lineage (H9-Osteo). In Phase 3, H9-Odont were seeded on 17% EDTA-treated dentine substrates in the presence of FGF-8 and BMP-4 for further differentiation. All experiments were performed in triplicate (n = 3). One-way anova was used to test hESC differentiation into different cell types. Post hoc Tukey's test was used to compare between groups. P < 0.05 was considered statistically significant.
H9-Odont expressed the odontoblastic marker DSPP gene 125.47 ± 0.1 (SD)-folds higher compared with H9-MSCs at mRNA level (real-time RT-PCR). Additionally, the flow cytometry results revealed 53.1 ± 3.4 (SD) % of DSP (+) cells in H9-Odont. Alternatively, H9-Osteo expressed 5.9 ± 2.2 (SD) % of DSP (+) cells. Moreover, the SEM results demonstrated that H9-Odont were found to undergo morphological changes from a fibroblast-like shape into more rounded shapes with cytoplasmic extensions into the dentinal tubules when seeded on 17% EDTA-treated dentine substrate in the presence of FGF-8 and BMP-4. However, H9-Osteo and H9-MSCs did not show similar morphological changes under similar culture milieu.
This study supports the potential of hESCs as a stable, consistent, unlimited and 'off-the-shelf' cell source to obtain odontoblastic cells for future clinical and research applications.
International Endodontic Journal 08/2013; 47(4). DOI:10.1111/iej.12150 · 2.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Excessive bone marrow adipocytes formation is tightly associated with development of osteoporosis. Considering the high heterogeneity of bone marrow stromal cells (BMSCs), identification of bone marrow adipocyte progenitors (BMAPs) within heterogeneous BMSCs may provide better cellular models for researches concerning osteoporosis development and therapy. However, currently there is no efficient method or specific surface makers available for BMAPs isolation. In the current study, we developed a novel BMAPs isolation method based on silica microbeads incubation and subsequent centrifugation in ficoll-paque. The "Sca-1+CD73-CD90-CD105+" subpopulation selected by this method exhibited significantly stronger adipogenic potential than non-selected BMSCs in vitro and could homogeneously differentiate to mature adipocytes within 4 days. Moreover, these cells also highly expressed a series of adipogenesis related genes even before differentiation. After long-term culture, however, BMAPs would gradually lose high adipogenic ability, but sorting CD105+ cells from BMAPs in later passages was able to retrieve the highly adipogenic subpopulation. In conclusion, this study demonstrated that BMAPs subpopulation could be effectively isolated from heterogeneous BMSCs by a special silica microbeads incubation method and re-enriched by sorting CD105+ cells. These findings offer convenient and repeatable approaches to obtain pure BMAPs for researches concerning pathogenic mechanisms and therapeutics development of osteoporosis.
Stem cells and development 06/2013; 22(18). DOI:10.1089/scd.2013.0199 · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human embryonic stem cells (hESCs) are uniquely endowed with a capacity for both self-renewal and multi-lineage differentiation. The aim of this investigation was to determine if short periods of cyclic mechanical strain enhanced dexamethasone, ascorbic acid and β-glycerophosphate (triple supplement) induced osteogenesis and bone nodule formation by hESCs. Colonies were cultured for 21 days and divided into control (no stretch) and three treatment groups; these were subjected to in-plane deformation of 2% for 5 sec (0.2 Hertz) every 60 sec for 1 hr on alternate days in BioFlex plates linked to a Flexercell strain unit over the following periods (Day 7-13), (D15-21) and (D7-21). Numerous bone nodules were formed which stained positively for osteocalcin and type I collagen; in addition, MTS assays for cell number as well as total collagen assays showed a significant increase in the D7-13 group compared to controls and other treatment groups. Alizarin red staining further showed that cyclic mechanical stretching significantly increased nodule size and mineral density between days 7-13 compared to control cultures and the other two experimental groups. We then performed a real time-PCR microarray on the D7-13 treatment group to identify mechanoresponsive osteogenic genes. Up-regulated genes included the transcription factors RUNX2 and SOX9, bone morphogenetic proteins BMP1, BMP4, BMP5, BMP6, transforming growth factor-β family members TGFB1, TGFB2 and TGFB3, and three genes involved in mineralization - ALPL, BGLAP and VDR. In conclusion, this investigation has demonstrated that four one hour episodes of cyclic mechanical strain acted synergistically with triple supplement to enhance osteogenesis and bone nodule formation by cultured hESCs. This suggests the development of methods to engineer 3-dimensional constructs of mineralized bone in vitro, could offer an alternative approach to osseous regeneration by producing a biomaterial capable of providing stable surfaces for osteoblasts to synthesize new bone, while at the same time able to be resorbed by osteoclastic activity - in other words, one that can recapitulate the remodelling dynamics of naturally occurring bone matrix.
Tissue Engineering Part A 04/2013; 19(19-20). DOI:10.1089/ten.TEA.2012.0308 · 4.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Periodontal ligament (PDL) cells in stationary two-dimensional culture systems are in a double default state. Our aim therefore was to engineer and characterize three-dimensional constructs, by seeding PDL cells into hyaluronan–gelatin hydrogel films (80–100 μm) in a format capable of being mechanically deformed.
Human PDL constructs were cultured with and without connective tissue growth factor (CTGF) and fibroblast growth factor (FGF)-2 in (i) stationary cultures, and (ii) mechanically active cultures subjected to cyclic strains of 12% at 0.2 Hz each min, 6 h/d, in a Flexercell FX-4000 Strain Unit. The following parameters were measured: cell number and viability by laser scanning confocal microscopy; cell proliferation with the MTS assay; the expression of a panel of 18 genes using real-time RT-PCR; matrix metalloproteinases (MMPs) 1–3, TIMP-1, CTGF and FGF-2 protein levels in supernatants from mechanically activated cultures with Enzyme-linked immunosorbent assays. Constructs from stationary cultures were also examined by scanning electron microscopy and immunostained for actin and vinculin.
Although initially randomly distributed, the cells became organized into a bilayer by day 7; apoptotic cells remained constant at approximately 5% of the total. CTGF/FGF-2 stimulated cell proliferation in stationary cultures, but relative quantity values suggested modest effects on gene expression. Two transcription factors (RUNX2 and PPARG), two collagens (COL1A1, COL3A1), four MMPs (MMP-1–3, TIMP-1), TGFB1, RANKL, OPG and P4HB were detected by gel electrophoresis and Ct values < 35. In mechanically active cultures, with the exception of P4HB, TGFB1 and RANKL, each was upregulated at some point in the time scale, as was the synthesis of MMPs and TIMP-1. SOX9, MYOD, SP7, BMP2, BGLAP or COL2A1 were not detected in either stationary or mechanically active cultures.
Three-dimensional tissue constructs provide additional complexity to monolayer culture systems, and suggest some of the assumptions regarding cell growth, differentiation and matrix turnover based on two-dimensional cultures may not apply to cells in three-dimensional matrices. Primarily developed as a transitional in vitro model for studying cell–cell and cell–matrix interactions in tooth support, the system is also suitable for investigating the pathogenesis of periodontal diseases, and importantly from the clinical point of view, in a mechanically active environment.
Journal of Periodontal Research 04/2013; 48(6). DOI:10.1111/jre.12072 · 2.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The innate immune response (IMR) is critical for the oral mucosa due to their continuous exposure to various oral pathogens. Keratinocytes play important role in IMR. Therefore, to date, keratinocytes from different sources have been used as in vitro research model for the study of IMR. However, current keratinocyte research models are hampered by the limited supply, patients' dependency and batch to batch variation. Therefore, in this study, we demonstrated the use of human embryonic stem cells (hESCs) derived keratinocytes (H9-Kert) as an alternative research model for the study of IMR.
The expression kinetics of toll-like receptor (TLR) 2, TLR 4, interleukin (IL) -6, IL-8, inducible nitric oxide synthase (iNOS) and tumour necrosis factor-alpha (TNF-α), in H9-Kert and immortalized human keratinocyte cell line (HaCaT) were analysed at mRNA levels by both reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR. The activation of the inflammatory transcription factor nuclear factor kappa-b (NFĸB) was assayed in these cells by transiently transfecting the cells with NFĸB reporter plasmid. Activation of NFĸB following treatment with heat-killed Porphyromonas gingivalis (P. gingivalis), an oral pathogen, was determined by assaying for the reporter, secreted alkaline phosphatase activity.
The expression of TLRs, cytokines and activation of NFĸB following bacterial stimulation showed in both H9-Kert and the widely used HaCaT keratinocyte cell line was similar.
Overall, our results support the potential application of hESCs as an alternative limitless cell source for primary keratinocytes which can be used as consistent and dependable research tool with minimum variations and no donor's dependency.
Journal of Oral Pathology and Medicine 03/2013; 42(8). DOI:10.1111/jop.12054 · 1.93 Impact Factor