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

Histology. Fractured tibiae were harvested at 7, 14, and 21 days. The tissues were fixed in 10% formalin for 24 hr and decalcified in 10% EDTA at pH 7.2 for 2 weeks. Samples were then processed, embedded in paraffin, and cut in 3-[micro]m sagittal sections. Three contiguous sections (100 [micro]m apart) for each specimen were stained with Alcian blue and counterstained with hematoxylin, orange G, and eosin (ABH/OG) as described previously (Zhang et al. 2002). Additional contiguous sections were stained for TRAP activity using naphthol AS-BI phosphate and counterstained with hematoxylin (Sigma).

Fracture histomorphometry. Quantitative analysis was performed on the sagittal ABH/OG- and TRAP-stained sections by histomorphometric analysis using Osteometrics software. Bone and cartilage formation was quantified on ABH/OG-stained slides by outlining the perimeter of the fracture callus under the 2x objective. Areas of new woven bone and cartilage were then traced. This procedure was repeated until the entire fracture callus had been evaluated. The areas of woven bone, cartilage, and total fracture callus were obtained directly in square millimeters from the software. The amount of mesenchymal tissue was then calculated by subtracting bone and cartilage area from the total callus area. Bone, cartilage, and mesenchymal tissue areas each were expressed as a percentage of total fracture callus. Osteoclast quantification was performed on the TRAP-stained slides. Using the 10x objective, the perimeter of fracture callus was outlined and individual TRAP positive osteoclasts were identified and counted. The entire fracture callus was evaluated. The number of TRA[P.sup. ] osteoclasts was then expressed per total area of fracture callus.

In situ hybridization. Plasmids corresponding to osteocalcin, collagen type II (Col II), and collagen type X (Col X) were used to synthesize [sup.35]S-labeled sense and anti-sense riboprobes as described previously (Ferguson et al. 1999; Zhang et al. 2002). Cut sections were incubated in hybridization buffer (50% formamide, 0.3 M NaCl, 20 mM Tris HCl, 5 mM EDTA, 10% dextran sulfate, 0.02% Ficoll, 0.02% bovine serum albumin, 0.02% polyvinyl pyrrolidone, and 0.5 mg/mL yeast RNA) containing each riboprobe at 10,000 cpm/[micro]L, and hybridization was performed at 55[degrees]C overnight. Nonspecifically bound probe was hydrolyzed with RNase A (20 [micro]g/[micro]L) and washed at high stringency at 55[degrees]C with 2x salt sodium citrate/50% formamide (Ferguson et al. 1999). Emulsion-dipped slides were exposed to beta emissions for 14 days.

Statistics. Data are expressed as mean [ or -] SEM. Statistical significance was determined using the Student t-test and analysis of variance (ANOVA) where appropriate. A value of p [less than or equal to] 0.05 was accepted as statistically significant.

Results

Pb exposure and whole-blood/bone Pb level determination. We established a reproducible murine Pb dosing protocol by introducing various concentrations of Pb into the drinking water of group A animals and determining BPb levels, similar to our rat exposure protocol (Cory-Slechta et al. 1997). We noted that BPb levels are quite variable after short-term exposure (1-2 weeks), whereas treatments from 3 to 6 weeks resulted in stable levels (Figure 1). To assure that this treatment resulted in soft and hard tissue exposure, we determined the Pb content in blood and bone (Table 1). These data indicate that oral dosing leads to environmentally relevant exposures (10-40 [micro]g/dL) and that BPb measurements faithfully reflect organ exposure in vivo. Additionally, mice appear to be very tolerant to these Pb exposures. No gross physical or behavioral changes were noted in animals with BPb levels three to four times greater than a lethal human exposure, in preliminary experiments. There was no statistically significant difference by ANOVA testing in body weight among the various dose groups in unpublished preliminary experiments (data not shown).