Changes in vasoactive factors associated with altered vessel morphology in the tibial metaphysis during ovariectomy-induced bone loss in rats.

We hypothesized that estrogen deficiency induces changes in bone vascularization which might be involved in bone loss mechanisms. First, we studied gene expression of angiogenic (vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2)) and vasodilator (endothelial nitric oxide synthase (ecNOS), neuronal NOS (nNOS), inducible NOS (iNOS), PTH-related protein (PTHrP), and its receptor PTH/PTHrP) factors in proximal tibial metaphysis of ovariectomized (OVX) rats and OVX 17beta-estradiol-treated rats at 3, 7, and 14 days. We then evaluated bone and vessel histomorphometry in secondary spongiosae by infusing vessels with a mixture of India ink/barium sulfate after 7 and 14 days of OVX. After 7 days expression of angiogenic and vasodilator factors decreased, concomitant with a decrease in the bone vessel number and possibly area. After 14 days all factors except FGF-2 exhibited either increased or normalized expression, which was associated with the stimulation of both bone formation and resorption. 17beta-Estradiol administration for 7 or 14 days prevented not only the OVX-induced changes in bone remodeling but also the morphological alterations observed in bone vessels. It also prevented the alterations in the expression of genes modified by OVX, except for that of FGF-2 whose transcription was similarly down-regulated in OVX rats with or without estrogen treatment.

Relationships between trabecular bone remodeling and bone vascularization: a quantitative study.

Beside its well-known role in bone development, vascularization plays a major role in bone cell migration for bone remodeling and metastatic tumor invasion. However, the various techniques used to identify vessels in bone have never been tested for trabecular bone vessel quantification, whereas bone remodeling quantitative parameters are commonly assessed. In this context, we developed and compared various histological techniques used to visualize blood vessels in rat bone in order to quantify them. First, several products were tested by intracardiac infusion to opacify the bone vascular network. The best results were obtained using either an India ink-1% agarose solution or an India ink-saturated barium sulfate solution followed by X-ray microradiography. Second, to identify the types of vessels, we also performed histoenzymology and immunohistochemistry stainings. Neither alkaline phosphatase (for endothelial cells) nor adenosine triphosphatase (ATPase) stainings (for smooth muscle cells) provided a low enough background to allow for vessel identification and quantification. For immunohistochemistry, various specific vessel constituents were analyzed: laminin, smooth muscle cell alpha-actin, factor VIII, and lectin Griffonia simplifolia. Anti-laminin and anti-smooth muscle cell alpha-actin antibodies gave the best results for quantification. Third, after optimization of these techniques, we performed quantitative bone and vessel histomorphometry on two groups of 12 rats each, for which bone remodeling and vessel number and area parameters were measured. No statistical differences were observed between the two groups, confirming the reproducibility of our measurements. A significant relationship was found between vessel number and histodynamic parameters; that is, bone formation rate correlated positively with India ink-positive vessel area (p < 0.009, r2 = 0.54) and alpha-actin-positive vessel number (p < 0.05, r2 = 0.66). Furthermore, we report reproducible techniques for visualization and quantification of vessels in bone that also allowed for simultaneous conventional bone histomorphometry. This methodology should help researchers to better understand the functional and anatomical relationship between trabecular bone and its vascularization during normal or pathological processes.