Large-scale time-lapse microscopy of Oct4 expression in human embryonic stem cell colonies.

Identification and quantification of the characteristics of stem cell preparations is critical for understanding stem cell biology and for the development and manufacturing of stem cell based therapies. We have developed image analysis and visualization software that allows effective use of time-lapse microscopy to provide spatial and dynamic information from large numbers of human embryonic stem cell colonies. To achieve statistically relevant sampling, we examined >680 colonies from 3 different preparations of cells over 5days each, generating a total experimental dataset of 0.9 terabyte (TB). The 0.5 Giga-pixel images at each time point were represented by multi-resolution pyramids and visualized using the Deep Zoom Javascript library extended to support viewing Giga-pixel images over time and extracting data on individual colonies. We present a methodology that enables quantification of variations in nominally-identical preparations and between colonies, correlation of colony characteristics with Oct4 expression, and identification of rare events.

Expression of cell adhesion and differentiation related genes in MC3T3 osteoblasts plated on titanium alloys: role of surface properties.

It is important to understand the cellular and molecular events that take place at the cell-material interface of implants used for bone repair. An understanding of the mechanisms involved in the initial stages of osteoblast interactions with the surface of the implant material is fundamental in deciding the fate of the cells that come in contact with it. In this study, we compared the relative gene expression of markers that are known to be associated with cell adhesion and differentiation in MC3T3 osteoblast cells, at various time points after plating the cells on surfaces of titanium (Ti) and its two alloys, titanium-zirconium (TiZr) and titanium-niobium (TiNb) by using Quantitative Real Time Polymerase Chain Reaction (RT-PCR). Our analysis indicated that expression of adhesion supporting genes was higher on TiZr surface as compared to Ti and TiNb. The behavior of these genes is possibly driven by a higher surface energy of TiZr. However no significant difference in the expression of differentiation related genes could be seen between the two alloys, although on both substrates it was higher as compared to unalloyed Ti. We propose that substrate composition of the alloys can influence the adhesion and differentiation related gene expression and that Ti alloys are better substrates for inducing osteogenesis as compared to unalloyed Ti.

Cell biological responses of osteoblasts on anodized nanotubular surface of a titanium-zirconium alloy.

Anodization of titanium and its alloys, under controlled conditions, generates a nanotubular architecture on the material surface. The biological consequences of such changes are poorly understood, and therefore, we have analyzed the cellular and molecular responses of osteoblasts that were plated on nanotubular anodized surface of a titanium-zirconium (TiZr) alloy. Upon comparing these results with those obtained on acid etched and polished surfaces of the same alloy, we observed a significant increase in adhesion and proliferation of cells on anodized surfaces as compared to acid etched or polished surface. The expression of genes related to cell adhesion was high only on anodized TiZr, but that of genes related to osteoblast differentiation and osteocalcin protein and extracellular matrix secretion were higher on both anodized and acid etched surfaces. Examination of surface morphology, topography, roughness, surface area and wettability using scanning electron microscopy, atomic force microscopy, and contact angle goniometry, showed that higher surface area, hydrophilicity, and nanoscale roughness of nanotubular TiZr surfaces, which were generated specifically by the anodization process, could strongly enhance the adhesion and proliferation of osteoblasts. We propose that biological properties of known bioactive titanium alloys can be further enhanced by generating nanotubular surfaces using anodization.

The influence of surface energy of titanium-zirconium alloy on osteoblast cell functions in vitro.

The success of an implant used for bone regeneration and repair is determined by the events that take place at the cell-material interface. An understanding of these interactions in vitro gives insights into the formulation of ideal conditions for their effective functioning in vivo. Thus, it is not only important to understand the physico-chemical properties of the materials but, also necessary to assess the cellular responses to them to determine their long-term stability and efficacy as implants. In the present study, we have compared the physico-chemical and biological properties of titanium (Ti) and two Ti-based alloys, namely: Ti- Zirconium (TiZr) and Ti-Niobium (TiNb). The morphology, chemical analysis, surface roughness, and contact angle measurements of the alloys were assessed by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), profilometer, and contact angle goniometer, respectively whereas the biological properties of the materials were evaluated by measuring the adhesion, proliferation, and differentiation of MC3T3-E1 osteoblast cells on the surfaces of these alloys. Our results indicate that the biological properties of osteoblasts were better on TiZr surface than on TiNb surface. Furthermore, the surface energy and substrate composition influenced the superior biological activity of the TiZr alloy.