The use of pimonidazole to characterise hypoxia in the internal environment of an in vivo tissue engineering chamber

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Summary

The distribution of hypoxic cells in an in vivo tissue engineering chamber was investigated up to 28 days post-implantation.

Methods

Arteriovenous loops were constructed and placed into bi-valved polycarbonate chambers containing 2×106 rat fibroblasts in basement membrane gel (BM gel). Chambers were inserted subcutaneously in the groin of male rats and harvested at 3 (n=6), 7 (n=6), 14 (n=4) or 28 (n=4) days. Ninety minutes before harvest, pimonidazole (60 mg/kg) was injected intraperitoneally. Chamber tissue was removed, immersion fixed, paraffin embedded, sectioned and stained immunohistochemically using hypoxyprobe-1 Mab that detects reduced pimonidazole adducts forming in cells, where pO2<10 mmHg.

Results

At 3 days a fibrin clot/BM gel framework filled the chamber. Seeded fibroblasts had largely died. The majority of 3 day chambers did not demonstrate tissue growth from the AV loop nor was pimonidazole binding present in these chambers. In one chamber in which tissue growth had occurred strong pimonidazole binding was evident within the new tissue. In four out of six 7 day chambers a broader proliferative zone existed extending up to 0.4 mm (approximately) from the AV loop endothelium which demonstrated intense pimonidazole binding. The two remaining 7 day chambers displayed even greater tissue growth (leading edge>0.7 mm from the AV loop endothelium), but very weak or no pimonidazole binding. At 14 and 28 days the fibrin/BM gel matrix was replaced by mature vascularised connective tissue that did not bind pimonidazole.

Conclusion

Employing a tissue engineering chamber, new tissue growth extending up to 0.4 mm from the AV loop endothelium (chambers≤7 days) demonstrated intense pimonidazole binding and, therefore, hypoxia. Tissue growth greater than 0.5 mm from the AV loop endothelium (7–28 days chambers) did not exhibit pimonidazole binding due to a significant increase in the number of new blood vessels and was, therefore, adequately oxygenated.

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.

References (18)

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