Intermittent Hydrostatic Pressurization Modulates Gene Expression in Human Dermal Fibroblasts Seeded in Three-Dimensional Polymer Scaffolds

2002 ◽  
Author(s):  
Steven B. Nicoll ◽  
Robert L. Mauck ◽  
Rick C. Tsay ◽  
Clark T. Hung ◽  
Gerard A. Ateshian
Keyword(s):  
Gene Expression ◽  
Hydrostatic Pressure ◽  
Cellular Response ◽  
Three Dimensional ◽  
Dermal Fibroblasts ◽  
Mechanical Stimuli ◽  
Human Dermal Fibroblasts ◽  
Polymer Scaffolds ◽  
Heat Shock Protein Expression ◽  
Disc Cells

Mechanical stimuli are known to regulate the morphology and differentiated function of connective tissue cells. In particular, hydrostatic pressure has been reported to alter cytoskeletal organization in osteoblast-like cells (1) and chondrocytes (2), and to modulate metabolic activity in both chondrocytes (3–5) and intervertebral disc cells (6). The cellular response to continuous hydrostatic pressure is generally catabolic (3) while intermittent hydrostatic pressure at frequencies ranging from 0.25–1.0 Hz (3–5) is anabolic, giving rise to increased expression and biosynthesis of extracellular matrix (ECM) components. Previously, human dermal fibroblasts in monolayer culture were shown to respond to hydrostatic pressure by increasing heat shock protein expression levels (7). In this study, we characterize the effects of intermittent hydrostatic pressure on gene expression in human dermal fibroblasts seeded in three-dimensional polymer scaffolds.

A New Approach to Cartilage Tissue Engineering Using Human Dermal Fibroblasts Seeded on Three-Dimensional Polymer Scaffolds

1998 ◽  
Vol 530 ◽  
Author(s):  
S. B. Nicoll ◽  
A. Wedrychowska ◽  
N. R. Smith ◽  
R. S. Bhatnagar
Keyword(s):  
Tissue Engineering ◽  
Lactic Acid ◽  
Articular Cartilage ◽  
Cartilage Tissue Engineering ◽  
Three Dimensional ◽  
Dermal Fibroblasts ◽  
High Density ◽  
Cartilage Tissue ◽  
Human Dermal Fibroblasts ◽  
Polymer Scaffolds

AbstractCurrent methods for correcting articular cartilage defects are limited by a scarcity of cartilage cells. Here we describe a novel method for the conversion of human dermal fibroblasts to chondrocyte-like cells and the potential application of this methodology to cartilage tissue engineering. Human neonatal foreskin fibroblasts were seeded on two-dimensional, tissue culture polystyrene (TCPS) in high density micromass cultures in the presence of staurosporine (50-200 nM), a protein kinase C (PKC) inhibitor, and lactic acid (40 mM) to induce functional hypoxia. Dermal fibroblasts were similarly cultured on three-dimensional polymer scaffolds composed of a non-woven polyglycolic acid (PGA) fiber mesh reinforced in a dilute solution of poly(L-lactic acid) (PLLA). At 24 hours, northern analysis revealed a staurosporine dose-dependent increase in aggrecan core protein expression in lactate-treated micromass cultures on TCPS, while type I collagen gene expression was virtually abolished in all cultures supplemented with staurosporine. The cells in these cultures displayed a rounded, cobblestone-shaped morphology typical of differentiated chondrocytes (most pronounced at 200 n.M staurosporine and 40 mM lactate), and were organized into nodules which stained positively with Alcian blue. When seeded on PGA/PLLA matrices under identical conditions as described for TCPS, a chondrocyte-like morphology was observed in cultures treated with lactate and staurosporine in contrast to the flattened sheets of fibroblast-like cells seen in untreated controls. Taken together, the above findings suggest that staurosporine treatment coupled with high density micromass culture in the presence of lactate induces chondrogenic differentiation in human dermal fibroblasts, and that these cells may be used in concert with three-dimensional polymer scaffolds for the repair of articular cartilage lesions.

Calcium pantothenate modulates gene expression in proliferating human dermal fibroblasts

2009 ◽  
Vol 18 (11) ◽  
pp. 969-978 ◽  
Author(s):  
Tonio Wiederholt ◽  
Ruth Heise ◽  
Claudia Skazik ◽  
Yvonne Marquardt ◽  
Sylvia Joussen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dermal Fibroblasts ◽  
Human Dermal Fibroblasts ◽  
Calcium Pantothenate

scholarly journals Validation of in vitro assays in three-dimensional human dermal constructs

2018 ◽  
Vol 41 (11) ◽  
pp. 779-788 ◽  
Author(s):  
Ayesha Idrees ◽  
Valeria Chiono ◽  
Gianluca Ciardelli ◽  
Siegfried Shah ◽  
Richard Viebahn ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Dermal Fibroblasts ◽  
In Vitro Assays ◽  
Dimensional System ◽  
Type I ◽  
Human Dermal Fibroblasts ◽  
Cell Viability Assay ◽  
Normal Human ◽  
Normal Human Dermal Fibroblasts

Three-dimensional cell culture systems are urgently needed for cytocompatibility testing of biomaterials. This work aimed at the development of three-dimensional in vitro dermal skin models and their optimization for cytocompatibility evaluation. Initially “murine in vitro dermal construct” based on L929 cells was generated, leading to the development of “human in vitro dermal construct” consisting of normal human dermal fibroblasts in rat tail tendon collagen type I. To assess the viability of the cells, different assays CellTiter-Blue®, RealTime-Glo™ MT, and CellTiter-Glo® (Promega) were evaluated to optimize the best-suited assay to the respective cell type and three-dimensional system. Z-stack imaging (Live/Dead and Phalloidin/DAPI-Promokine) was performed to visualize normal human dermal fibroblasts inside matrix revealing filopodia-like morphology and a uniform distribution of normal human dermal fibroblasts in matrix. CellTiter-Glo was found to be the optimal cell viability assay among those analyzed. CellTiter-Blue reagent affected the cell morphology of normal human dermal fibroblasts (unlike L929), suggesting an interference with cell biological activity, resulting in less reliable viability data. On the other hand, RealTime-Glo provided a linear signal only with a very low cell density, which made this assay unsuitable for this system. CellTiter-Glo adapted to three-dimensional dermal construct by optimizing the “shaking time” to enhance the reagent penetration and maximum adenosine triphosphate release, indicating 2.4 times higher viability value by shaking for 60 min than for 5 min. In addition, viability results showed that cells were viable inside the matrix. This model would be further advanced with more layers of skin to make a full thickness model.

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