Toxicity due to Pb exposure remains a major public health concern and presents a broad spectrum of pathologies in children and adults. Over the last decade, our group has focused on the effects of Pb on bone and cartilage and its potential role in osteoporosis (Campbell et al. 2004
; Puzas et al. 2004
). Because the clinical manifestation of osteoporosis is fracture and because fracture healing is a proven model for examining cellular and molecular aspects of skeletal repair, we evaluated the effects of in vivo
Pb exposure on callus formation, maturation, and remodeling. In addition to osteoporosis, Pb exposure is known to contribute to dental caries (Moss et al. 1999
) and complications of skeletal growth in children (Kafourou et al. 1997
) via poorly defined mechanisms. As such, elucidation of the Pb effects on angio-genesis, stem cell recruitment, endochondral ossification, osteoclastogenesis, and bone remodeling during fracture healing could also have implications for these other human conditions. Additionally, individuals who have high-injury-risk occupations and are concurrently exposed to Pb may also be susceptible to fracture nonunions.
Our first aim was to develop a reproducible dosing regimen that could achieve stable BPb levels over time. For this study we chose < 3, 15, and 40 μg/dL in whole blood as our targets of human background, intermediate, and high Pb exposures, respectively. We found a tight correlation between the drinking water concentration and BPb levels () that was similar to our observations in rats (Cory-Slechta et al. 1997
). We verified the incorporation of Pb into hard and soft tissues (), which revealed similar concentrations to those previously reported in bone (Pounds et al. 1991
). Of interest, no gross toxic effects of very high BPb exposures (~ 400 μg/dL) were noted. Because this is three to four times the lethal dose in humans, future studies using murine models with human extrapolation may need to consider higher dosing.
In this study, remarkable effects of Pb on fracture healing were clearly apparent, even at the lowest dose (–). The phenotype can be described as an increase in chondrogenesis and delay in endochondral maturation, vascular invasion, and resorption. Although fracture healing is a highly ordered biologic process that requires precise temporal and spatial regulation, it is known that various compensatory mechanisms have evolved to rescue healing under adverse conditions. In our experience with various drug treatments and genetically deficient strains, essentially all closed stable fractures heal in mice (Flick et al. 2003
; Zhang et al. 2002
). Thus, the finding that the profound Pb-induced phenotype on day 14 is completely resolved by day 21 is not surprising. However, the presence of fibrous nonunions in three out of four mice with very high Pb exposure is remarkable given that all other mice studied (n
> 100) healed. Because it is well known that mice have an extremely robust ability to heal fractures, we speculate that mice healed fractures at Pb exposures at which humans could not.
Although the precise mechanism by which Pb inhibits fracture-healing remains a topic for future investigation, the phenotype is very reproducible and is somewhat reminiscent of phenotypes described in other mouse models. The increased chondrogenesis observed is similar to that seen in the fracture callus of parathyroid hormone and prostaglandin-treated mice, indicating that Pb may be an agonist of protein kinase A signaling in chondrocytes, as predicted in our in vitro
studies (Zuscik et al. 2002
). The literature provides three potential explanations for the persistence of cartilage in the fracture callus. The first is a defect in chondrocyte apoptosis, as seen in mice with defective tumor necrosis factor receptors (Gerstenfeld et al. 2001
). The second is a defect in vascular invasion of the cartilage, as seen in the matrix metalloproteinase-9 knockout mice (Colnot et al. 2003
). The third is the inhibition of mesenchymal stem cell differentiation into osteoprogenitors, as seen in mice deficient in cyclooxygenase-2 (Zhang et al. 2002
), which is required for callus mineralization. Evidence for this mechanism is supported by our finding that Pb-treated animals have a significant decrease in osteoprogenitor frequency (). An additional possibility is inhibition of osteoclastic resorption and remodeling of the fracture callus, as seen in mice with defective M-CSF and RANKL signaling. However, our findings that in situ
osteoclast numbers (, ) and OCP frequency () are unaffected by in vivo
Pb exposure renders this possibility less likely.
The overall clinical significance of Pb inhibition of fracture healing relates to persons with osteoporosis. We have argued that because of the high environmental Pb exposures from the 1940s to 1960s, women currently going through menopause are at an additional risk of osteoporosis (Puzas et al. 2004
). It is now well recognized that factors released from bone during resorption, such as TGF-β, can act on cells in the bone marrow to induce the production of osteoclastic stimulating factors or to inhibit osteoblastic new bone formation (Evans et al. 1989
; Yin et al. 1999
). As a consequence of the high bone turnover, which would release Pb from its inactive state in bone hydroxyapatite crystals, an additional imbalance of bone resorption over formation would occur from Pb’s preferential toxic effects on osteoblasts. Our results indicate that osteoporotic, Pb-exposed patients may sustain a fragility fracture earlier and heal their fractures at a slower rate compared with non-Pb-exposed osteoporotic individuals. Future investigations into the molecular mechanisms of Pb effects on osteoporosis and fracture healing are warranted.