Technical Reference #2029

2029.

Biocompatibility of a synthetic extracellular matrix on immortalized vocal fold fibroblasts in 3-D culture Xia Chen; Susan L. Thibeault, University of Wisconsin Madison, Acta Biomaterialia, 6(2029), (2010)
Link To Paper

Abstract:
In order to promote wound repair and induce tissue regeneration an engineered hyaluronan (HA) hydrogel – Carbylan GSX which contains di(thiopropionyl) bishydrazide-modified hyaluronic acid di(thiopropionyl) bishydrazide-modified gelatin and polyethylene glycol diacrylate – has been developed for extracellular matrix (ECM) defects of the superficial and middle layers of the lamina propria. The purpose of this study was to evaluate the biocompatibility of Carbylan GSX in a previously established immortalized human vocal fold fibroblast (hVFF) cell line prior to human clinical trials. Immortalized hVFF proliferation viability apoptosis and transcript analysis for both ECM constituents and inflammatory markers were measured for two-dimensional and three-dimensional (3-D) culture conditions. There were no significant differences in morphology cell marker protein expression proliferation viability and apoptosis of hVFF cultured with Carbylan GSX compared to Matrigel a commercial 3-D control after 1 week. Gene expression levels for fibromodulin transforming growth factor-beta1 and tumor necrosis factor-alpha were similar between Carbylan GSX and Matrigel. Fibronectin hyaluronidase 1 and cyclooxygenase II expression levels were induced by Carbylan GSX whereas interleukins 6 and 8 Col I and hyaluronic acid synthase 3 expression levels were decreased by Carbylan GSX. This investigation demonstrates that Carbylan GSX may serve as a natural biomaterial for tissue-engineering of human vocal folds.

Keywords:
Human vocal fold fibroblasts Three-dimensional culture Extracellular matrix HA hydrogel (Carbylan GSX) Cell proliferation

Materials & Methods:
For 2-D culture 6-well plates (MatTek Ashland MA) were seeded with immortalized hVFFs at 0.5 106 wellÿ1 on transwell permeable supports (inserts with 0.4 lm membrane pore size; Millipore Inc. Billerica MA) with 500 ll of the gelated hydrogel Carbylan GSX. For 3-D culture a single cell suspension was mixed with Carbylan GSX or Matrigel solutions at a final concentration of 2 106 cells mlÿ1 by gentle stirring then 500 ll of this mixture was placed in each well of a 6-well plate with the inserts. After gelation (gel thickness was approximately 0.5 mm) cell culture medium (DMEM–10% FBS) was added above and below gel. The media was exchanged every 3 days.

Microscopic Technique
Confocal microscopy, Inverted confocal microscopy

Cell Type(s)
primary hVFF cells