PMID- 17430089
OWN - NLM
STAT- MEDLINE
DCOM- 20070712
LR  - 20220129
IS  - 1076-3279 (Print)
IS  - 1076-3279 (Linking)
VI  - 13
IP  - 5
DP  - 2007 May
TI  - Real-time detection of stress in 3D tissue-engineered constructs using NF-kappaB 
      activation in transiently transfected human dermal fibroblast cells.
PG  - 1013-24
AB  - The main objective of this study was to develop a nondestructive reporter system 
      for assessing the response of human cells contained within a three-dimensional 
      (3D) tissue-engineered construct to exogenous stress. Dermal fibroblasts were 
      transiently transfected with a reporter construct linked to nuclear factor kappaB 
      (NF-kappaB) activation which led to expression of a nonstable form of enhanced 
      green fluorescent protein (d2EGFP) after stimulation. This led to a temporary 
      production of fluorescence, which could be readily detected but was not 
      intrinsically toxic, as cells were able to metabolize the initial cycle of d2EGFP 
      produced. This permitted the model to be used for restimulation post recovery. To 
      investigate the performance and predictive ability of this method for assessing 
      cellular response to stress in 3D, we used a range of compounds known to have 
      pro-inflammatory or oxidative properties. Tumor necrosis factor-alpha (TNF-alpha) 
      and interleukin-1-beta (IL-1beta) were selected for having a direct cytokine 
      action; lipopolysaccharide (LPS) was selected for modeling bacterial-mediated 
      inflammation; and hydrogen peroxide was selected as a crude method for delivering 
      an oxidative stress. Transfected cells were stimulated with the above compounds 
      in 3D and the synthesis of d2EGFP was detected as a measure of NF-kappaB 
      activation. The resultant fluorescence was scored using a series of 
      photomicrographs taken by epifluorescence microscopy. All agents activated 
      NF-kappaB when cells were grown in 3D scaffolds but did not cause any significant 
      reduction in cell viability as measured by a standard MTT-ESTA viability test. 
      Parallel NF-kappaB activation and MTT measurements was also conducted in 
      two-dimension (2D) and confirmed findings in 3D. The 3D model described using a 
      fluorescent reporter gene is a highly sensitive and reliable method for detecting 
      cellular stress and represents a key step in developing tissue engineering models 
      with the potential for screening pharmaceutical and cosmetic compounds, as an 
      alternative to existing in vitro and in vivo methods.
FAU - Canton, Irene
AU  - Canton I
AD  - Department of Engineering Materials, Kroto Research Institute, University of 
      Sheffield, and Northern General Hospital, Sheffield, United Kingdom.
FAU - Sarwar, Umran
AU  - Sarwar U
FAU - Kemp, E Helen
AU  - Kemp EH
FAU - Ryan, Anthony J
AU  - Ryan AJ
FAU - MacNeil, Sheila
AU  - MacNeil S
FAU - Haycock, John W
AU  - Haycock JW
LA  - eng
GR  - BB/D524983/1/BB_/Biotechnology and Biological Sciences Research Council/United 
      Kingdom
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Tissue Eng
JT  - Tissue engineering
JID - 9505538
RN  - 0 (Interleukin-1beta)
RN  - 0 (Lipopolysaccharides)
RN  - 0 (NF-kappa B)
RN  - 0 (Tumor Necrosis Factor-alpha)
RN  - 0 (enhanced green fluorescent protein)
RN  - 147336-22-9 (Green Fluorescent Proteins)
SB  - IM
MH  - Cell Culture Techniques
MH  - Cells, Cultured
MH  - Dermis/cytology/*metabolism
MH  - Drug Evaluation, Preclinical
MH  - Fibroblasts/cytology/*metabolism
MH  - Genes, Reporter
MH  - Green Fluorescent Proteins/biosynthesis/genetics
MH  - Humans
MH  - Inflammation/genetics/metabolism
MH  - Interleukin-1beta/pharmacology
MH  - Lipopolysaccharides/pharmacology
MH  - Microscopy, Fluorescence
MH  - *Models, Biological
MH  - NF-kappa B/genetics/*metabolism
MH  - *Oxidative Stress/drug effects
MH  - *Tissue Engineering
MH  - Transfection
MH  - Tumor Necrosis Factor-alpha/pharmacology
EDAT- 2007/04/14 09:00
MHDA- 2007/07/13 09:00
CRDT- 2007/04/14 09:00
PHST- 2007/04/14 09:00 [pubmed]
PHST- 2007/07/13 09:00 [medline]
PHST- 2007/04/14 09:00 [entrez]
AID - 10.1089/ten.2006.0357 [doi]
PST - ppublish
SO  - Tissue Eng. 2007 May;13(5):1013-24. doi: 10.1089/ten.2006.0357.