Biomedical subjects

Eric M Darling

Publications and source records attributed to Eric M Darling.

7 recordsLinked to original sources

PubMed2006

A thin-layer model for viscoelastic, stress-relaxation testing of cells using atomic force microscopy: do cell properties reflect metastatic potential?

Atomic force microscopy has rapidly become a valuable tool for quantifying the biophysical properties of single cells. The interpretation of atomic force microscopy-based indentation tests, however, is highly dependent on the use of an appropriate theoretical model of the testing configuration. In this study, a novel, thin-layer viscoelastic model for stress relaxation was developed to quantify the mechanical properties of chondrosarcoma cells in different configurations to examine the hypothesis that viscoelastic properties reflect the metastatic potential and invasiveness of the cell using three well-characterized human chondrosarcoma cell lines (JJ012, FS090, 105KC) that show increasing chondrocytic differentiation and decreasing malignancy, respectively. Single-cell stress relaxation tests were conducted at 2 h and 2 days after plating to determine cell mechanical properties in either spherical or spread morphologies and analyzed using the new theoretical model. At both time points, JJ012 cells had the lowest moduli of the cell lines examined, whereas FS090 typically had the highest. At 2 days, all cells showed an increase in stiffness and a decrease in apparent viscosity compared to the 2-h time point. Fluorescent labeling showed that the F-actin structure in spread cells was significantly different between FS090 cells and JJ012/105KC cells. Taken together with results of previous studies, these findings indicate that cell transformation and tumorigenicity are associated with a decrease in cell modulus and apparent viscosity, suggesting that cell mechanical properties may provide insight into the metastatic potential and invasiveness of a cell.

Actins↗

PubMed2005

Growth factor impact on articular cartilage subpopulations.

We have examined the effects of growth factor stimulation on superficial and growth zone chondrocyte populations. Zonal articular chondrocytes from 8-month-old Spanish goat distal femurs were plated in monolayer cultures and stimulated by using insulin-like growth factor I (IGF-I), basic fibroblast growth factor (bFGF), and transforming growth factor-beta1 (TGF-beta1). Gene expression for collagen I and II, aggrecan, and superficial zone protein were evaluated every week for 3 weeks. Finally, proteoglycan and collagen deposition were measured for each experimental group. Major differences existed in the behavior of superficial and growth zone chondrocytes, the most apparent being the higher capacity for protein synthesis by the growth zone population. Variations also existed regarding growth factor treatment. TGF-beta1 had the greatest effect on proliferation over 8 days. With respect to differentiation, IGF-I increased average collagen II gene expression in the growth zone populations in comparison with growth zone controls. IGF-I increased aggrecan gene expression for the same groups. Superficial zone populations exhibited lower collagen II, collagen I, and aggrecan gene expression than the growth zone populations under all conditions. However, superficial zone protein expression was dramatically elevated in superficial zone populations by TGF-beta1. Collagen I expression showed a general increase under all conditions compared with initial values. Combined biosynthesis results showed that the superficial populations secreted little to no collagen, especially collagen II, in comparison with their growth zone counterparts. Glycosaminoglycan production was also much lower than for the growth zone groups. TGF-beta1 and IGF-I increased collagen II production in the growth zone populations. TGF-beta1 increased glycosaminoglycan secretions in the superficial zone populations and in the growth zone populations, whereas IGF-I produced an increase in glycosaminoglycan secretion only in the growth zone populations. Thus, growth factors elicit different proliferation, gene expression, and biosynthesis responses from zonal chondrocyte subpopulations.

Animals↗

PubMed2005

Rapid phenotypic changes in passaged articular chondrocyte subpopulations.

Articular chondrocytes are often expanded in vitro and then used to assist in healing articular cartilage defects. We investigated the extent of dedifferentiation in monolayer-passaged, zonal articular chondrocytes by using quantitative, real-time PCR. The relative gene expressions for collagen type I and II, aggrecan, and superficial zone protein were analyzed for relevant passage numbers (P0-P4) to determine how the expansion of chondrocytes affects the expression of cartilage extracellular matrix proteins. Results reveal that dramatic changes occur as early as first passage. Furthermore, these changes are shown to persist even when the expanded cells are encapsulated in 3D, alginate beads. Successful tissue engineering and autologous cell transplantation procedures rely heavily on having a cell source that expresses the chondrocytic phenotype. The results of this study suggest that major problems exist at the front-end of cartilage regeneration efforts.

Aggrecans↗

PubMed2004

Zonal and topographical differences in articular cartilage gene expression.

Articular cartilage is composed of phenotypically different zones. In young articular cartilage, there are only two distinct zones: superficial and growth. The zones have different mechanical properties and play specific roles within functional cartilage tissue. In small animal models, it is difficult to separate the zones quickly and efficiently using only a dissecting microscope. Surface abrasion is a method that has been developed to harvest cells from articular cartilage to produce highly purified samples in a simple, reproducible process. Using this harvesting technique, the superficial zone has been separated from the underlying growth zone. Superficial cells comprised approximately 4% of the total cells obtained. Superficial and growth zone chondrocytes from articular cartilage were analyzed using real-time RT-PCR. Expressed superficial zone protein was 3-fold greater in the superficial zone population than in the growth zone population (p < 0.01). This, along with histological evidence, indicates that surface abrasion is successful as a zonal separation technique. Additionally, type II collagen was expressed 8-fold more abundantly in the growth zone than in the superficial zone (p < 0.005). There was no difference in aggrecan expression between the two zones. Regional variations among the femoral groove and medial and lateral condyles were also examined. No significant variations in SZP, type II collagen, or aggrecan were found, which makes the pooling of zonal cells from different regions an acceptable option for tissue engineering studies.

Animals↗

PubMed2003

Articular cartilage bioreactors and bioprocesses.

This review summarizes the major approaches for developing articular cartilage, using bioreactors and mechanical stimuli. Cartilage cells live in an environment heavily influenced by mechanical forces. The development of cartilaginous tissue is dependent on the environment that surrounds it, both in vivo and in vitro. Chondrocytes must be cultured in a way that gives them the proper concentration of nutrients and oxygen while removing wastes. A mechanical force must also be applied during the culturing process to produce a phenotypically correct tissue. Four main types of forces are currently used in cartilage-culturing processes: hydrostatic pressure, direct compression, "high"-shear fluid environments, and "low"-shear fluid environments. All these forces have been integrated into culturing devices that serve as bioreactors for articular cartilage. The strengths and weaknesses of each device and stimulus are explored, as is the future of cartilage bioreactors.

Animals↗

PubMed2003

Biomechanical strategies for articular cartilage regeneration.

Two major contributions to the development of articular cartilage are growth factors and mechanical loading. Growth factors have long been used to modulate the secretion of certain molecules from different cells. The TGF-beta superfamily, specifically the BMPs, CDMPs, OPs, and GDFs, have a dramatic effect on the development of bone and cartilage tissue. These growth factors help produce an extracellular matrix that can withstand extreme loading conditions in the body. In addition to growth factors, it is known that mechanical forces stimulate the synthesis of extracellular proteins in vitro and in vivo and can affect the tissue's overall structure. Load-bearing tissue, such as articular cartilage, will atrophy in the absence of mechanical forces, and this observation has caused researchers to incorporate mechanical stimulation into the tissue engineering process. This article focuses on the importance of mechanical forces in tissue engineering of articular cartilage and the growth factors that help stimulate the formation of load-bearing tissue.

Cartilage, Articular↗

PubMedDate not supplied

Retaining zonal chondrocyte phenotype by means of novel growth environments.

The loss of phenotype in articular chondrocytes expanded in monolayer has been established as a possible contributor to the deficiencies associated with in vitro cartilage engineering and autologous cell transplantation procedures. We cultured zonal articular chondrocytes on tissue culture plastic, collagen II-coated polystyrene, and aggrecan-coated polystyrene in an effort to find a surface that can either prevent or slow the loss of phenotype. In addition, we encapsulated passaged cells in agarose to examine the effect of three-dimensional culture on redifferentiating zonal chondrocytes. We used real-time polymerase chain reaction to measure the relative gene expression levels of collagen I and II, aggrecan, and superficial zone protein over relevant passages (P0-P4). Results showed that tissue culture plastic and the collagen II-coated surface induced rapid loss of phenotype in zonal articular chondrocytes. The aggrecan-coated surface had a less detrimental effect on the chondrocytic phenotype of seeded cells, inducing gene expression characteristics comparable to those of agarose-encapsulated cells. Furthermore, when chondrocytes that had been previously passaged on a collagen II surface were placed on an aggrecan surface, the zonal cells showed a dramatic change in gene expression from fibroblastic to chondrocytic. These results indicate that a culture environment using aggrecan as a substratum or agarose as a scaffold is crucial to the development of phenotypically correct articular cartilage.

Aggrecans↗