scholarly journals Accelerated construction of an in vitro model of human periodontal ligament tissue: vacuum plasma combined with fibronectin coating and a polydimethylsiloxane matrix

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7036 ◽  
Author(s):  
Wen Liao ◽  
Yoshiya Hashimoto ◽  
Yoshitomo Honda ◽  
Peiqi Li ◽  
Yang Yao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Plasma Treatment ◽  
Periodontal Ligament ◽  
Orthodontic Treatment ◽  
Cellular Proliferation ◽  
Cellular Adhesion ◽  
Cyclic Stretch ◽  
Vacuum Plasma ◽  
Fibronectin Coating

Tying shape memory wires to crowded teeth causes the wires to deform according to the dental arch. This deformation results in a resilient force that is delivered to the tooth. The appropriate amount of force can activate the osteogenetic and osteoclastic ability of the periodontal ligament (PDL) and the tooth can be moved. This is the biological basis of orthodontic treatment. To achieve further insight into the mechanisms underlying orthodontic treatment, we examined whether accelerated construction of an in vitro human PDL fibroblast (HPdLF) stretching model can be achieved by combining fibronectin coating and vacuum plasma treatment with polydimethylsiloxane (PDMS) cell-culture chambers. Each chamber was randomly assigned to a no-surface modification (NN), fibronectin coating (FN), vacuum plasma treatment (PN), or vacuum plasma treatment followed by a fibronectin coating (PF) treatment protocol. The physical and chemical features and ability to promote cellular proliferation of the PDMS chamber surfaces were evaluated. Cellular adhesion of four materials were evaluated and two best-proliferated groups were considered as better model-constructing surfaces and used in subsequent experiments and used in subsequent experiments. HPdLFs were cultured on these two kinds of chambers without stretching for 3 days, then with stretching for 7 days. Time-course gene expression cellular morphology were evaluated. Chambers in the PN group had high wettability and surface component changes. The FN and PF chambers had high cellular proliferation ability. They were selected into subsequent experiments. After 3 days of culturing HPdLFs on the PF and PN chambers, the cells in the PF chambers had significantly higher levels of runt-related transcription factor 2 (Runx-2) and osteocalcin (OCN) gene expression compared with the cells in the PN chambers. After cyclic stretch application to the cells in the PN and PF chambers, expression of the type-3 collagen (COL-3) gene in PF group continued to increase for 7 days and was significantly higher than that in the PN group from day 5 onwards. The HPdLFs in the PF group showed parallel alignment from days 3 to 7 after imposition of cyclic stretch, while those in the PN group aligned in parallel from day 5 on. Our results suggested that applying a fibronectin coating to a PDMS chamber after plasma treatment can accelerate establishment of an in vitro PDL stretching model.

scholarly journals Interleukin-1β Induced Matrix Metalloproteinase Expression in Human Periodontal Ligament-Derived Mesenchymal Stromal Cells under In Vitro Simulated Static Orthodontic Forces

2021 ◽  
Vol 22 (3) ◽  
pp. 1027
Author(s):  
Christian Behm ◽  
Michael Nemec ◽  
Alice Blufstein ◽  
Maria Schubert ◽  
Xiaohui Rausch-Fan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Periodontal Ligament ◽  
Induced Expression ◽  
Gene And Protein Expression ◽  
Tensile Strains ◽  
Orthodontic Forces ◽  
Low Magnitude

The periodontal ligament (PDL) responds to applied orthodontic forces by extracellular matrix (ECM) remodeling, in which human periodontal ligament-derived mesenchymal stromal cells (hPDL-MSCs) are largely involved by producing matrix metalloproteinases (MMPs) and their local inhibitors (TIMPs). Apart from orthodontic forces, the synthesis of MMPs and TIMPs is influenced by the aseptic inflammation occurring during orthodontic treatment. Interleukin (IL)-1β is one of the most abundant inflammatory mediators in this process and crucially affects the expression of MMPs and TIMPs in the presence of cyclic low-magnitude orthodontic tensile forces. In this study we aimed to investigate, for the first time, how IL-1β induced expression of MMPs, TIMPs and how IL-1β in hPDL-MSCs was changed after applying in vitro low-magnitude orthodontic tensile strains in a static application mode. Hence, primary hPDL-MSCs were stimulated with IL-1β in combination with static tensile strains (STS) with 6% elongation. After 6- and 24 h, MMP-1, MMP-2, TIMP-1 and IL-1β expression levels were measured. STS alone had no influence on the basal expression of investigated target genes, whereas IL-1β caused increased expression of these genes. In combination, they increased the gene and protein expression of MMP-1 and the gene expression of MMP-2 after 24 h. After 6 h, STS reduced IL-1β-induced MMP-1 synthesis and MMP-2 gene expression. IL-1β-induced TIMP-1 gene expression was decreased by STS after 6- and 24-h. At both time points, the IL-1β-induced gene expression of IL-1β was increased. Additionally, this study showed that fetal bovine serum (FBS) caused an overall suppression of IL-1β-induced expression of MMP-1, MMP-2 and TIMP-1. Further, it caused lower or opposite effects of STS on IL-1β-induced expression. These observations suggest that low-magnitude orthodontic tensile strains may favor a more inflammatory and destructive response of hPDL-MSCs when using a static application form and that this response is highly influenced by the presence of FBS in vitro.

Expression of MMP-8 and MMP-13 Genes in the Periodontal Ligament during Tooth Movement in Rats

2003 ◽  
Vol 82 (8) ◽  
pp. 646-651 ◽  
Author(s):  
I. Takahashi ◽  
M. Nishimura ◽  
K. Onodera ◽  
J.-W. Bae ◽  
H. Mitani ◽  
...  
Keyword(s):  
Gene Expression ◽  
Periodontal Ligament ◽  
Tooth Movement ◽  
Orthodontic Tooth Movement ◽  
Polymerase Chain Reaction Analysis ◽  
Tension And Compression ◽  
Polymerase Chain

Periodontal ligament tissue is remodeled on both the tension and compression sides of moving teeth during orthodontic tooth movement. The present study was designed to clarify the hypothesis that the expression of MMP-8 and MMP-13 mRNA is promoted during the remodeling of periodontal ligament tissue in orthodontic tooth movement. We used the in situ hybridization method and semi-quantitative reverse-transcription/polymerase chain-reaction analysis to elucidate the gene expression of MMP-8 and MMP-13 mRNA. Expression of MMP-8 and MMP-13 mRNA transiently increased on both the compression and tension sides during active tooth movement in vivo. The gene expression of MMP-8 and MMP-13 was induced by tension, while compression indirectly promoted the gene expression of MMP-8 and MMP-13 through soluble factors in vitro. Thus, we concluded that the expression of MMP-8 and MMP-13 is differentially regulated by tension and compression, and plays an important role in the remodeling of the periodontal ligament.

scholarly journals The Impact of Bioactive Surfaces in the Early Stages of Osseointegration: An In Vitro Comparative Study Evaluating the HAnano® and SLActive® Super Hydrophilic Surfaces

2020 ◽  
Vol 2020 ◽  
pp. 1-11
Author(s):  
Rodrigo A. da Silva ◽  
Geórgia da Silva Feltran ◽  
Marcel Rodrigues Ferreira ◽  
Patrícia Fretes Wood ◽  
Fabio Bezerra ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Adhesion ◽  
Cell Cycle Progression ◽  
Cellular Adhesion ◽  
Biological Behavior ◽  
Early Stages ◽  
Genes Encoding ◽  
Bioactive Surfaces ◽  
The Impact

There is an increased effort on developing novel and active surfaces in order to accelerate their osteointegration, such as nanosized crystalline hydroxyapatite coating (HAnano®). To better understand the biological behavior of osteoblasts grown on HAnano® surface, the set of data was compared with SLActive®, a hydrophilic sandblasted titanium surface. Methodologically, osteoblasts were seeded on both surfaces up to 72 hours, to allow evaluating cell adhesion, viability, and set of genes encoding proteins related with adhesion, proliferation, and differentiation. Our data shows HAnano® displays an interesting substrate to support cell adhesion with typical spread morphologic cells, while SLActive®-adhering cells presented fusiform morphology. Our data shows that the cellular adhesion mechanism was accompanied with upexpression of integrin β1, Fak, and Src, favoring the assembling of focal adhesion platforms and coupling cell cycle progression (upmodulating of Cdk2, Cdk4, and Cdk6 genes) in response to HAnano®. Additionally, both bioactive surfaces promoted osteoblast differentiation stimulus, by activating Runx2, Osterix, and Alp genes. Although both surfaces promoted Rankl gene expression, Opg gene expression was higher in SLActive® and this difference reflected on the Rankl/Opg ratio. Finally, Caspase1 gene was significantly upmodulated in response to HAnano® and it suggests an involvement of the inflammasome complex. Collectively, this study provides enough evidences to support that the nanohydroxyapatite-coated surface provides the necessary microenvironment to drive osteoblast performance on dental implants and these stages of osteogenesis are expected during the early stages of osseointegration.

scholarly journals G9a drives hypoxia-mediated gene repression for breast cancer cell survival and tumorigenesis

2017 ◽  
Vol 114 (27) ◽  
pp. 7077-7082 ◽  
Author(s):  
Francesco Casciello ◽  
Fares Al-Ejeh ◽  
Greg Kelly ◽  
Donal J. Brennan ◽  
Shin Foong Ngiow ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Tumor Growth ◽  
Cellular Proliferation ◽  
Gene Signature ◽  
Breast Cancer Patients ◽  
Hypoxic Response ◽  
And Migration

G9a is an epigenetic regulator that methylates H3K9, generally causing repression of gene expression, and participates in diverse cellular functions. G9a is genetically deregulated in a variety of tumor types and can silence tumor suppressor genes and, therefore, is important for carcinogenesis. Although hypoxia is recognized to be an adverse factor in tumor growth and metastasis, the role of G9a in regulating gene expression in hypoxia has not been described extensively. Here, we show that G9a protein stability is increased in hypoxia via reduced proline hydroxylation and, hence, inefficient degradation by the proteasome. This inefficiency leads to an increase in H3K9me2 at its target promoters. Blocking the methyltransferase activity of G9a inhibited cellular proliferation and migration in vitro and tumor growth in vivo. Furthermore, an increased level of G9a is a crucial factor in mediating the hypoxic response by down-regulating the expression of specific genes, includingARNTL,CEACAM7,GATA2,HHEX,KLRG1, andOGN. This down-regulation can be rescued by a small molecule inhibitor of G9a. Based on the hypothesis that the changes in gene expression would influence patient outcomes, we have developed a prognostic G9a-suppressed gene signature that can stratify breast cancer patients. Together, our findings provide an insight into the role G9a plays as an epigenetic mediator of hypoxic response, which can be used as a diagnostic marker, and proposes G9a as a therapeutic target for solid cancers.

scholarly journals A bilayered tissue engineered in vitro model simulating the tooth periodontium

2021 ◽  
Vol 42 ◽  
pp. 232-245
Author(s):  
A Khadre ◽  
ELM Raif ◽  
S Junaid ◽  
OM Goudouri ◽  
W Refaat ◽  
...  
Keyword(s):  
Gene Expression ◽  
Periodontal Ligament ◽  
Alveolar Bone ◽  
Cell Attachment ◽  
Human Tooth ◽  
Periodontal Ligament Cells ◽  
Collagen Membrane ◽  
Tissue Component

Due to the complexity of the structure of the tooth periodontium, regeneration of the full tooth attachment is not a trivial task. There is also a gap in models that can represent human tooth attachment in vitro and in vivo. The aim of this study was to develop a bilayered in vitro construct that simulated the tooth periodontal ligament and attached alveolar bone, for the purpose of tissue regeneration and investigation of physiological and orthodontic loading. Two types of materials were used to develop this construct: sol-gel 60S10Mg derived scaffold, representing the hard tissue component of the periodontium, and commercially available Geistlich Bio-Gide® collagen membrane, representing the soft tissue component of the tooth attachment. Each scaffold was dynamically seeded with human periodontal ligament cells (HPDLCs). Scaffolds were either cultured separately, or combined in a bilayered construct, for 2 weeks. Characterisation of the individual scaffolds and the bilayered constructs included biological characterisation (cell viability, scanning electron microscopy to confirm cell attachment, gene expression of periodontium regeneration markers), and mechanical characterisation of scaffolds and constructs. HPDLCs enjoyed a biocompatible 3-dimensional environment within the bilayered construct components. There was no drop in cellular gene expression in the bilayered construct, compared to the separate scaffolds.

Abstract 311: TGFb/Smad3 Stimulation Drives Smooth Muscle Cell De-differentiation

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Daniel M DiRenzo ◽  
Xudong Shi ◽  
Stephen Seedial ◽  
Lian-wang Guo ◽  
Bo Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Smooth Muscle ◽  
Balloon Angioplasty ◽  
Intimal Hyperplasia ◽  
Cellular Proliferation ◽  
Smooth Muscle Actin ◽  
Tgfβ Signaling ◽  
And Migration ◽  
Proliferation And Migration

Restenosis, or vessel re-narrowing, occurs in approximately 25-50% of arterial interventions involving balloon angioplasty due to the formation of a proliferative plaque in the vessel lumen termed neo-intimal hyperplasia. Arterial smooth muscle cells (SMCs) contribute to neo-intimal hyperplasia through a de-differentiation process that includes downregulation of their contractile gene expression and conversion to a phenotype that includes proliferation, migration, and matrix synthesis. Expression of TGFβ and its downstream signaling protein, Smad3, are greatly upregulated following vascular injury, including balloon angioplasty. Classically, TGFβ signaling has been shown to suppress SMC proliferation and migration in vitro, however, Smad3 overexpressing SMCs demonstrate enhanced proliferation and migration. Furthermore, overexpression of Smad3 in rat carotid arteries enhances neo-intimal hyperplasia following balloon angioplasty. These results lead us to hypothesize that TGFβ signaling, in the context of upregulated Smad3, drives SMC de-differentiation leading to enhanced cellular proliferation and migration. We utilized primary rat SMCs infected with adenovirus constructs overexpressing Smad3 or GFP control and performed gene expression microarrays 24 hours following TGFβ administration. We observed statistically significant (p<0.05) upregulation of 145 genes and downregulation of 76 genes by more than 3-fold. GO term analysis revealed that genes involved in embryonic tissue development (41 genes) and stem/progenitor cell differentiation (27 genes) were significantly enriched in TGFβ/Smad3 stimulated cells. Confirmatory qRT-PCR demonstrated that the contractile genes SM-MHC, smooth muscle actin, and calponin were significantly downregulated -6.3, -2.7 and -2.1 fold, respectively. In contrast, stem/developmental related genes Cxcr4, Cd34, Wnt11, Wnt2b and IL11 were significantly upregulated by 105.2, 22.3, 11.5, 14.0, and 12.5 fold, respectively. These results strongly suggest that TGFβ/Smad3 stimulation is a powerful de-differentiation signal in SMCs and plays an important role in the development of neo-intimal hyperplasia.

Abstract 310: Role of Cardiac Fibroblast in Cardioprotective Effects of Prior Transient ACE Inhibition

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Taben M Hale ◽  
Lauren A Biwer ◽  
Karen M D’Souza
Keyword(s):  
Gene Expression ◽  
Cellular Proliferation ◽  
Cardiac Fibroblasts ◽  
Cardiac Injury ◽  
Ace Inhibition ◽  
Cardiac Fibroblast ◽  
Heart Weight ◽  
Nitric Oxide Synthase Inhibitor

Prior treatment with the ACE inhibitor enalapril followed by washout protects against nitric oxide synthase inhibitor (L-NAME) induced fibrosis, cellular proliferation, and cardiac dysfunction. The present study investigated i) whether in vivo L-NAME administration induces a change in cardiac fibroblast phenotype that persists in vitro, ii) whether prior ACE inhibition protects against L-NAME induced changes in cardiac fibroblasts. SHR were divided into 3 groups: Control, L-NAME (C+L: 7d), enalapril+L-NAME (E+L: 14d enalapril + 14d washout + 7d L-NAME). MAP was measured by radiotelemetry (n=5-9), injury assessed by histology (n=6-10), and heart weight to body weight (HW/BW) was determined after 0 or 7 days of L-NAME in C+L and E+L (n=6-10). In separate rats cardiac fibroblasts were isolated after 7 days of L-NAME (C+L, E+L) or placebo (Con) and cultured to passage 1 (n=10-12). Gene expression was measured by quantitative real-time PCR. L-NAME increased MAP in C+L (22±4.1%) and E+L (21±3.6%) rats. Prior enalapril induced a persistent 13% reduction in HW/BW. L-NAME increased heart mass in E+L (7%) but not C+L; however, HW/BW remained 8% lower than C+L at sacrifice. L-NAME induced infarct in 70% of C+L and 40% of E+L hearts. Cardiac fibroblasts demonstrated a significant increase in proliferation rate in C+L, but not E+L, relative to control (C+L: 1.75-fold vs. con; E+L 1.09-fold vs. con). Fibroblasts from C+L hearts tended to have increased Collagen I and III gene expression. Despite hypertension, cardiac injury, and increased HW/BW; fibroblasts isolated from E+L proliferated at the same rate as those from control. In contrast, those isolated from C+L were hyperproliferative with a tendency toward increased capacity for collagen production. It may be that the fibroblast phenotype from E+L hearts would protect against infarct expansion and account, in part, for the previously reported cardioprotection in these rats.

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