The use of pimonidazole to characterise hypoxia in the internal environment of an in vivo tissue engineering chamber
Section snippets
The AV loop chamber model—surgical method
Full approval for these experiments was obtained from the Animal Ethics Committee of St Vincent's Hospital Melbourne, Australia. All experiments were conducted according to National Health and Medical Research Council (Australia) guidelines.
A custom-made polycarbonate chamber, in the shape of a flat cylinder with a removable lid and an access hole for the pedicle, external diameter 14 mm, height 4 mm, was manufactured in the Department of Chemical and Biomolecular Engineering, University of
Fibroblast cultures
The mean absorbance for MTT in fibroblast cultures under hypoxic and normoxic conditions was found to be almost identical (data not shown) indicating cell death did not occur in hypoxic cells over a 240 min time frame.
Hypoxyprobe-1 labeling was strongly evident in cultured hypoxic fibroblasts after 240 min suggesting that fibroblasts are capable of incorporating pimonidazole (Fig. 2(A)). Normoxic cells in identical culture conditions did not incorporate pimonidazole (Fig. 2(B)).
Three days
Three days post-AV
Discussion
Tissue growth proceeded in the chamber over a period of 2 weeks before becoming stabilised. This is consistent with our previous findings in this model.4, 6 In contrast to our previous studies9 we were not able to demonstrate survival of seeded fibroblasts in the chamber. BM gel (not used previously) was the carrier for the fibroblasts. It is a mouse product and would, therefore, elicit an immune response. The fibroblasts were derived from an inbred stain of rats, but may also have elicited a
Acknowledgements
Grant funding from the National Health and Medical Research Council (Australia), the L. E. W. Carty Charitable Fund and the Transport Accident Commission Victoria is gratefully acknowledged. Dr S. O. P. Hofer was funded by the Dutch Cancer Foundation (K. W. F.), Amsterdam, Netherlands. Surgical assistance from staff at the Experimental Medical and Surgical Unit, St Vincent's Hospital, is gratefully acknowledged.
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2018, Trends in BiotechnologyCitation Excerpt :The gradient of hypoxia formed within the chamber due to the separation of the AVL from surrounding tissues has also been shown to be angio-inductive. Areas that are initially hypoxic such as the entrance to the chamber and the venous segments correlate with greatest angiogenesis [39,40]. Sprouting from the AVL occurs predominantly from the venous portion initially [18,40,41], and apart from hypoxia this has been postulated to be due to the thinner wall of a vein compared with the arterial segment.
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