Lead exposure inhibits fracture healing and is associated with increased chondrogenesis, delay in cartilage mineralization, and a decrease in osteoprogenitor frequency

Environmental Health Perspectives, June, 2005 by Jonathan J. Carmouche, J. Edward Puzas, Xinping Zhang, Prarop Tiyapatanaputi, Deborah A. Cory-Slechta, Robert Gelein, Michael Zuscik, Randy N. Rosier, Brendan F. Boyce, Regis J. O'Keefe, Edward M. Schwarz

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Pb inhibits fracture healing. By 14 days after fracture, all animals showed radiographic evidence of fracture callus formation (Figure 2). However, radiographs of Pb-exposed animals demonstrated a marked increase in radiolucency within the fracture callus compared with controls (Figure 2D-F). At 21 days, all groups showed evidence of remodeling of the fracture callus with no remarkable difference in the three groups (Figure 2G-I).

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Consistent with our radiographic findings, histologic analysis revealed no remarkable differences at 7 and 21 days because all fracture sites consisted of undifferentiated mesenchyme and fibrocartilage at the early time point and remodeling bone at the latter time point (Figure 3). In our day 14 untreated animals, we see the normal pattern of newly formed bone through a cartilage intermediate, as evidenced by the areas of Alcian blue--stained cartilage throughout the callus (Figure 3D). However, in the 14-day Pb-treated groups, immature cartilage accounts for the large radiolucency identified by X ray (Figure 3E-F). Histomorphometry of the day 14 fracture calluses showed a significant delay in endochondral ossification because the Pb-treated mice had a 4- to 5-fold increase in unmineralized cartilage with a commensurate decrease in bone (Figure 4). Interestingly, there was a nonlinear response to the effects of Pb on bone tissue because a similar effect was seen in both the 55 and 230 ppm treatment groups.

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In situ hybridization and TRAP staining in the 14-day fracture group helped assess phenotypic gene expression and quantify osteoclasts in the fracture callus, respectively (Figure 5). Robust expression of Col II, decreased expression of Col X, and absence of both the mature osteoblast marker osteocalcin and TRA[P.sup. ] osteoclasts were noted in Pb-treated animals. This confirmed the prevalence of immature cartilage in the fracture callus. Importantly, gene expression outside of this immature cartilage was indistinguishable from untreated controls (Figure 5A-I), as was osteoclast number (Figures 4 and 5J-L). Thus, low Pb exposures did not completely inhibit any process of fracture healing. Rather, Pb delayed endochondral ossification.

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A cohort of group A mice (n = 4) were exposed to 2,300 ppm Pb and analyzed radiographically and histologically. In contrast to the lower-dose treatment groups (group B), the radiolucency in the day-14 X rays was not accompanied by surrounding fracture callus (Figure 6A). Furthermore, histology failed to identify evidence of endochondral ossification at the fracture site. Day 21 specimens confirmed fibrous nonunions in 75% of the group A 2,300 ppm animals (Figure 6B-C). Thus, Pb can completely inhibit fracture healing at very high doses.

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The direct effects of Pb on chondrocytes (Zuscik et al. 2002), osteoclasts (Bonucci et al. 1983), and osteoblasts (Klein and Wiren 1993; Long et al. 1990; Pounds et al. 1991) have been previously documented. Our experiments on cells isolated from Pb-exposed animals focus on the mechanism by which Pb inhibits fracture healing by determining its effects on progenitor cells. When osteoprogenitor cell differentiation was examined using a bone nodule formation assay, von Kossa staining revealed that Pb-treated animals produced significantly fewer nodules at both the 55 ppm and 230 ppm dosing regimens (Figure 7). Because there was no Pb exposure in the culture medium during osteoblast differentiation, we interpret these results as a reduction in the total number of osteoprogenitor cells.