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

Fracture healing is a complex orchestration of several cell types. It is unique in that healing occurs by reformation of bone rather than scar tissue. Fracture healing involves primary recruitment, proliferation, and differentiation of both osteoprogenitors (osteoblast progenitors), for intramembranous ossification, and undifferentiated mesenchymal cells, for endochondral bone formation (Barnes et al. 1999; Einhorn 1998). Fracture healing is an established means of studying the formation and repair of skeletal elements in vivo as well as the crucial signaling pathways involved (Zhang et al. 2002).

Given the known effects on cells involved in bone formation and remodeling and the absence of literature on Pb in skeletal repair, the purpose of the present study was to evaluate the effects of elevated blood Pb levels on fracture healing. We employed an established murine tibia fracture model (Bonnarens and Einhorn 1984; Einhorn 1998) to characterize the effects of Pb on skeletal repair.

Materials and Methods

Pb exposure and whole blood Pb level determination. All Pb solutions were made using Pb acetate (Gibco, Grand Island, NY) dissolved in distilled water. All mice had unrestricted access to the normal rodent diet supplied by the vivarium staff, and drinking water was replenished at least one time per week by the investigative staff to ensure continuous Pb exposure. All Pb waste solutions were disposed of appropriately. Mice were housed in groups of [less than or equal to] animals per microisolator cage in the vivarium and maintained on a 12-hr light/dark cycle. All procedures were carried out in accordance with the regulations of and following the approval of the University of Rochester Animal Use and Care Committee.

Female C57/B16 mice (n = 32) at 6 weeks of age were divided into eight groups of four mice each. Each group was exposed to one of the following doses: 0, 55, 230, 580, 1,160, 1,750, 2,300, or 5,800 ppm Pb in the drinking water. This preliminary exposure was carried out to determine the whole-blood Pb (BPb) concentrations needed to approximate environmentally relevant levels of human exposure. This cohort is referred to as group A. Pb exposures of 0, 55, and 230 ppm Pb in the drinking water were selected as most environmentally relevant. A second cohort (group B) of female C57/B16 mice (n = 54) was divided into three groups of 18 animals and housed under conditions as outlined above. All experiments related to the analysis of the fracture healing process were performed with group B except one, which documented chronic nonunions in mice exposed to 2,300 ppm Pb.

Before Pb exposure, 100 [micro]L whole blood samples were collected for baseline BPb level measurement. Samples were collected, using the saphenous vein technique (Hem et al. 1998), into 100 [micro]L nitric acid-washed volumetric capillary tubes (VWR, Buffalo Grove, IL). Each whole-blood sample was then immediately diluted 1:10 with a matrix modifier solution containing 0.2% N[H.sub.4][H.sub.2]P[O.sub.4], 0.5% Triton X-100, and 0.2% HN[O.sub.3] (Parsons 1993). Whole blood was collected for BPb analysis at 3-week intervals.