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

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.