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

Osteoclastogenesis. Splenocytes were seeded at 1.75 x [10.sup.5] cells/well in a 96-well plate in [alpha]-MEM supplemented with macrophage-colony-stimulating factor (M-CSF; 30 ng/mL) and receptor activator nuclear factor [kappa]B ligand (RANKL; 100 ng/mL). Fifty percent medium was added the next day, and medium was changed every other day thereafter. Cultures were incubated at 37[degrees]C for 6-7 days and then fixed and stained for tartrate-resistant acid phosphatase (TRAP) using the leukocyte acid phosphatase kit (Sigma). The number of [TRAP.sup. ] multinucleated cells was then counted to quantify osteoclast formation as described previously (Zhang et al. 2001).

Flow cytometry. Surface protein staining was performed on splenocytes. After erythrocyte lysis, a single-cell suspension was incubated in DMEM with 10% FBS. Cells were harvested in PBS containing 5 mM EDTA and stained for CD11b with biotin-labeled antibodies, as described previously (Li et al. 2004). Data were acquired using a FACScalibur cytometer and analyzed with Cell Quest software (Beckton Dickenson, Bedford, MA).

Macrophage colony-forming assay. The in vitro colony-forming assay was performed as described previously (Franzoso et al. 1997). Freshly isolated splenocytes were plated at [10.sup.5] cells/mL in a 35-mm dish. Splenocytes were cultured in methylcellulose-based medium (StemCell Technologies, Vancouver, British Columbia, Canada) supplemented with M-CSF (30 ng/mL) for 12 days. Individual colonies, defined as > 40 cells, were then quantified under an inverted microscope. The total number of colonies represents the original number of monocyte/macrophage and osteoclast precursors (Schwarz et al. 1997).

Bone resorption assay. Splenocytes were seeded at 1.75 x [10.sup.5] cells/well on sterile 4 mm x 4 mm bovine femoral cortical bone wafers. Cells were cultured in [alpha]-MEM with 10% FBS supplemented with M-CSF (30 ng/mL), RANKL (100 ng/mL), 1% L-glutamine, 1% penicillin/streptomycin, and 1% nonessential amino acids (Gibco). Fifty percent medium was added the next day, and medium was changed every other day thereafter. After 12 days, the wafers were scraped, dried, stained with toluidine blue, and examined under a 40x objective. Wafer images were captured, and resorption pits on the wafer surface were traced to determine the total pitted area using Osteometrics software (Osteometrics, Atlanta, GA) as described previously (Childs et al. 2001).

Fracture. After 6 weeks of Pb exposure, mice were anesthetized by intraperitoneal injection of 100 mg/kg ketamine HCl and 15 mg/kg xylazine. After adequate sedation, the surgical site was prepared with 70% ethanol and an incision was made about the left knee. A 25-gauge 5/8-inch needle was inserted lateral to the patellar tendon and into the tibial marrow space. This needle was then removed and a 0.25-mm diameter insect pin (Fine Science Tools, Foster City, CA) was placed into the tibia. The pin was trimmed proximally at the level of the tibial plateau. The wound was then closed with 4-0 nylon sutures. The left tibiae were placed in a modification of the guillotine three point-bending device described by Bonnarens and Einhorn (1984) and Hiltunen et al. (1993). The tibial diaphyses were fractured, without trauma to the overlying skin, using a force exerted by a 540 g weight dropped 16.5 cm. Radiographs characterized the fracture and confirmed intramedullary fixation. Radiographs were obtained using a Faxitron cabinet X-ray system (Faxitron, Wheeling, IL). The radiographic exam was repeated at 7-day intervals to follow healing and confirm maintenance of fixation.