Biomedical subjects

Douglas W Hamilton

Publications and source records attributed to Douglas W Hamilton.

5 recordsLinked to original sources

PubMed2007

The effect of substratum topography on osteoblast adhesion mediated signal transduction and phosphorylation.

Substratum surface topography is a powerful modulator of cell behaviour, but how it influences intracellular signaling is largely unknown. We investigated the influence of microfabricated topographies on the activation of nonreceptor tyrosine kinases Src, FAK and ERK 1/2, as well as the transcription factor, Runx2, in rat osteoblasts, cultured on substrata that varied in their ability to promote bone-like tissue formation. Total tyrosine phosphorylation increased on grooves, tapered pits, and gap cornered boxes, relative to the levels found on smooth surfaces, with the greatest activity at 1 week. Src levels was higher on smooth than on any other surface, but FAK and ERK 1/2 phosphorylation were highest on groove and gap-cornered boxes up to 6 weeks. Inhibition of Src phosphorylation with PP2 inhibited FAK and ERK 1/2 phosphorylation on grooves, but had no detectable effect on either FAK or ERK 1/2 on smooth substratum. We suggest that osteoblast response to substrata with specific topographical features requires FAK-Y397-Src-Y416 complexes for ERK 1/2 phosphorylation, but on smooth surfaces, Src independent methods of ERK 1/2 activation are present.

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PubMed2007

A new experimental system for the extended application of cyclic hydrostatic pressure to cell culture.

Mechanical forces have been shown to be important stimuli for the determination and maintenance of cellular phenotype and function. Many cells are constantly exposed in vivo to cyclic pressure, shear stress, and/or strain. Therefore, the ability to study the effects of these stimuli in vitro is important for understanding how they contribute to both normal and pathologic states. While there exist commercial as well as custom-built devices for the extended application of cyclic strain and shear stress, very few cyclic pressure systems have been reported to apply stimulation longer than 48 h. However, pertinent responses of cells to mechanical stimulation may occur later than this. To address this limitation, we have designed a new cyclic hydrostatic pressure system based upon the following design variables: minimal size, stability of pressure and humidity, maximal accessibility, and versatility. Computational fluid dynamics (CFD) was utilized to predict the pressure and potential shear stress within the chamber during the first half of a 1.0 Hz duty cycle. To biologically validate our system, we tested the response of bone marrow progenitor cells (BMPCs) from Sprague Dawley rats to a cyclic pressure stimulation of 120/80 mm Hg, 1.0 Hz for 7 days. Cellular morphology was measured using Scion Image, and cellular proliferation was measured by counting nuclei in ten fields of view. CFD results showed a constant pressure across the length of the chamber and no shear stress developed at the base of the chamber where the cells are cultured. BMPCs from Sprague Dawley rats demonstrated a significant change in morphology versus controls by reducing their size and adopting a more rounded morphology. Furthermore, these cells increased their proliferation under cyclic hydrostatic pressure. We have demonstrated that our system imparts a single mechanical stimulus of cyclic hydrostatic pressure and is capable of at least 7 days of continuous operation without affecting cellular viability. Furthermore, we have shown for the first time that BMPCs respond to cyclic hydrostatic pressure by alterations in morphology and increased proliferation.

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PubMed2006

Biomimetic modification of titanium dental implant model surfaces using the RGDSP-peptide sequence: a cell morphology study.

Surface topography and (bio)chemistry are key factors in determining cell response to an implant. We investigated cell adhesion and spreading patterns of epithelial cells, fibroblasts and osteoblasts on biomimetically modified, smooth and rough titanium surfaces. The RGD bioactive peptide sequence was immobilized via a non-fouling poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) molecular assembly system, which allowed exploitation of specific cell-peptide interactions even in the presence of serum. As control surfaces, bare titanium and bio-inactive surfaces (scrambled RDG and unfunctionalized PLL-g-PEG) were used. Our findings demonstrated that surface topography and chemistry directly influenced the attachment and morphology of all cell types tested. In general, an increase in cell number and more spread cells were observed on bioactive substrates (containing RGD) compared to bio-inactive surfaces. More fibroblasts were present on smooth than on rough topographies, whereas for osteoblasts the opposite tendency was observed. Epithelial cell attachment did not follow any regular pattern. Footprint areas for all cell types were significantly reduced on rough compared to smooth surfaces. Osteoblast attachment and footprint areas increased with increasing RGD-peptide surface density. However, no synergy (interaction) between RGD-peptide surface density and surface topography was observed for osteoblasts neither in terms of attachment nor footprint area.

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PubMed2006

Chondrocyte aggregation on micrometric surface topography: a time-lapse study.

Cellular aggregation or mesenchymal "condensation" is a prerequisite in the process of chondrogenesis. It has been observed that during in vitro engineering of cartilage, chondrocytes form aggregates in the initial cell seeding process of polymer scaffolds. However, the exact mechanism behind this aggregation has yet to be elucidated, although cell collision has been implicated. As all polymers have a surface topography, we hypothesized that topography may play a role in chondrocyte aggregation. 1(st) and 2(nd) passage chondrocytes were seeded on micrometric topography ranging from 0.75 to 8 microm in depth and 5 to 12.5 microm in width. Both 1(st) and 2(nd) passage cells formed aggregates as cells collided, and larger aggregates formed as aggregates collided with each other on the grooves. Furthermore, aggregates migrated parallel to the direction of the groove long axis. F-actin organization was altered only in the cellular layer in direct contact with the surface; stress fibers oriented in the direction of the groove long axis. Chondrocytes maintained type II collagen expression on all groove depths. This study shows that micrometric grooves could be an effective means for studying chondrocyte aggregation, and could additionally be utilized in the regeneration of cartilage.

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PubMedDate not supplied

Characterization of the response of bone marrow-derived progenitor cells to cyclic strain: implications for vascular tissue-engineering applications.

One of the major failings in vascular tissue engineering is the limited capacity of autologous differentiated cells to reconstitute tissues. A logical solution is to use multipotent progenitor cells, which in vascular treatments have been underutilized. Although biochemical stimulation has been explored to differentiate bone marrow-derived progenitor cells (BMPCs) to smooth muscle cells (SMCs), the use of biomechanical forces in differentiation remains unexplored. The purpose of this work was to explore the effects of cyclic strain alone on BMPC morphology, proliferation, and differentiation. BMPCs were isolated from rat bone marrow and, after 7 days in culture, the cells grew in distinct multilayered colonies. BMPCs were stimulated with 10% strain at 1 Hz for 7 days. Observations showed that cyclic strain inhibited proliferation (p < 0.05) and caused alignment of the cells (p < 0.05) and of the F-actin cytoskeleton perpendicular to the direction of strain. In addition, cyclic strain resulted in expression by the cells of vascular smooth muscle alpha-actin and h1-calponin. This work demonstrates the potential of physiologic biomechanical stimulation in the differentiation of BMPCs to SMCs, and this could have important implications for vascular tissue engineering and other therapies in which cell sourcing is a major concern.

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