DIFFERENTIATING ANGIOGENESIS FROM VESSEL RECRUITMENT OF PERFUSED MICROVASCULATURE DURING BONE HEALING

H. Winet
[Orthopaedic Hospital/UCLA, Department of Orthopaedic Surgery]

INTRODUCTION Bone trauma usually initiates a healing cascade characterized by an inflammatory phase which is followed by repair, which includes angiogenesis. The speed of inflammation onset does not allow sufficient time for significant angiogenesis and the accompanying increase in perfused vasculature is achieved by recruitment of previously "resting" vessels. During the succeeding repair phase angiogenesis replaces recruitment as the cause of increased perfused vascularity. Differentiating the point at which the second process overtakes the first is difficult if the only criterion for both is vascularity. The same dilemma occurs when bone tissue is responding to an implanted biomaterial or chemical agent. A solution may lie in the fact that angiogenesis is usually focused in venular capillaries which are of the order of 8 µm in diameter while recruitment occurs in vessels with a wider diameter range. To test the hypothesis that such a discrimination was possible in vivo, healing bone was exposed to a known vessel irritant, nicotine.

METHODS & MATERIALS Right tibias of nine female NZW rabbits were implanted with optical bone chambers (BCIs) which they were allowed to osseointegrate for three weeks. Chamber ends were surgically exposed, then viewed under a fluorescence intravital microscope. Each rabbit was injected IV with 0.25gm fluorescein isothiocyanate dextran of 70 kDa molecular weight and its BCI bone ingrowth compartment photographed under fluorescence illumination (480nm). After the first observation, which established baseline, inflammation-linked (due to exposure surgery) vascularity, five rabbits were treated daily with a Habitrol nicotine patch which delivered 5.25 mg/day of the drug. The four control rabbits received a strip of isopore tape. The patch/tape was placed on the inner surface of the ear and changed every day. Observations were repeated weekly until the 12th week post-implantation of the BCIs (i.e. 9 weeks). Photographic images were scanned, digitized and measured using Image-1 software. All measures were normalized to the value obtained at the first observation. Vessel length and caliber (fluorescent diameter) were measured and results from each week were evaluated using one-way ANOVA (SYSTAT). Statistical significance was set at p £0.05.

RESULTS Perfused vascularity for controls followed the historical BCI pattern of increasing between weeks 4 and 6, after which it plateaued. In contrast, nicotine-treated rabbits showed no such increase in vascularity over the entire course. The differences became highly significant by the end of the study. Vessel caliber for controls showed a typical decrease in value to a plateau at week 7. Nicotine-treated vessel calibers did not significantly differ from controls at any point in the study. A trend in these vessels toward slightly smaller calibers is evident in the curve but this difference must be considered merely suggestive.

CONCLUSIONS & DISCUSSION The results obtained supported the hypothesis not by showing that vessel caliber was significantly different in the two experimental groups, while vascularity remained the same. Instead, they showed that even if angiogenesis occurs, there is a decrease in vessel perfusion in the presence of nicotine. That angiogenesis is occurring is supported by decrease in caliber for both groups. The failure of these new vessels to be perfused may be traced to the known constrictive effect of nicotine on vascular smooth muscle. Blood is probably flowing more slowly in the vessels observed. Of course, it is also possible that the decreased shear stress accompanying slower blood flow reduces the stimulus for bone capillaries to enlarge. If the latter case is true, vessels could be simply mimicking angiogenesis by maintaining small calibers. The problem warrants further study.