The Association between Environmental Lead Exposure and Bone Density in Children

Osteoporosis is a decrease in bone mineral density (BMD) that predisposes individuals to fractures. Although an elderly affliction, a predisposition may develop during adolescence if a sufficient peak BMD is not achieved. Rat studies have found that lead exposure is associated with decreased BMD. However, human studies are limited. We hypothesized that the BMD of children with high lead exposure would be lower than the BMD of children with low lead exposure. We collected data on 35 subjects; 16 had low cumulative lead exposure (mean, 6.5 μg/dL), and 19 had high exposure (mean, 23.6 μg/dL). All were African American; there was no difference between the groups by sex, age, body mass index, socioeconomic status, physical activity, or calcium intake. Significant differences in BMD between low and high cumulative lead exposure were noted in the head (1.589 vs. 1.721 g/cm(2)), third lumbar vertebra (0.761 vs. 0.819 g/cm(2)), and fourth lumbar vertebra (0.712 vs. 0.789 g/cm(2)). Contrary to our hypothesis, subjects with high lead exposure had a significantly higher BMD than did subjects with low lead exposure. This may reflect a true phenomenon because lead exposure has been reported to accelerate bony maturation by inhibiting the effects of parathyroid hormone–related peptide. Accelerated maturation of bone may ultimately result in a lower peak BMD being achieved in young adulthood, thus predisposing to osteoporosis in later life. Future studies need to investigate this proposed model

Analysis of Vertical Ground Reaction Force Variables during a Sit to Stand Task in Participants Recovering from a Hip Fracture

Background: A Sit to Stand task following a hip fracture may be achieved through compensations (e.g. bilateral arms and uninvolved lower extremity), not restoration of movement strategies of the involved lower extremity. The primary purpose was to compare upper and lower extremity movement strategies using the vertical ground reaction force during a Sit to Stand task in participants recovering from a hip fracture to control participants. The secondary purpose was to evaluate the correlation between vertical ground reaction force variables and validated functional measures. Methods: Twenty eight community dwelling older adults, 14 who had a hip fracture and 14 control participants completed the Sit to Stand task on an instrumented chair designed to measure vertical ground reaction force, performance based tests (Timed up and go, Berg Balance Scale and Gait Speed) and a self report Lower Extremity Measure. A MANOVA was used to compare functional scales and vertical ground reaction force variables between groups. Bivariate correlations were assessed using Pearson Product Moment correlations. Findings: The vertical ground reaction force variables showed significantly higher bilateral arm force, higher uninvolved side peak force and asymmetry between the involved and uninvolved sides for the participants recovering from a hip fracture (Wilks\u27 Lambda=3.16, P=0.019). Significant correlations existed between the vertical ground reaction force variables and validated functional measures. Interpretation: Participants recovering from a hip fracture compensated using their arms and the uninvolved side to perform a Sit to Stand. Lower extremity movement strategies captured during a Sit to Stand task were correlated to scales used to assess function, balance and falls risk

Influence of Upper Extremity Assistance on Lower Extremity Force Application Symmetry in Individuals Post–Hip Fracture During the Sit-to-Stand Task

STUDY DESIGN: Controlled laboratory study using a cross-sectional design. OBJECTIVES: To compare lower extremity force applications during a sit-to-stand (STS) task with and without upper extremity assistance in older individuals post–hip fracture to those of age matched controls. BACKGROUND: A recent study documented the dependence on upper extremity assistance and the uninvolved lower limb during an STS task in individuals post–hip fracture. This study extends this work by examining the effect of upper extremity assistance on symmetry of lower extremity force applications. METHODS: Twenty-eight community-dwelling elderly subjects, 14 who had recovered from a hip fracture and 14 controls, participated in the study. All participants were independent ambulators. Four force plates were used to determine lower extremity force applications during an STS task with and without upper extremity assistance. The summed vertical ground reaction forces (vGRFs) of both limbs were used to determine STS phases (preparation/rising). The lower extremity force applications were assessed statistically using analysis of variance models. RESULTS: During the preparation phase, sideto-side symmetry of the rate of force development was significantly lower for the hip fracture group for both STS tasks (P\u3c.001). During the rising phase, the vGRF impulse of the involved limb was significantly lower for the hip fracture group for both STS tasks (P = .045). The vGRF impulse for the uninvolved limb was significantly increased when participants with hip fracture did not use upper extremity assistance compared to elderly controls (P = .002). This resulted in a significantly lower vGRF symmetry for the hip fracture group during both STS tasks (P\u3c.001). CONCLUSION: Participants with hip fracture who were discharged from rehabilitative care demonstrated decreased side-to-side symmetry of lower extremity loading during an STS task, irrespective of whether upper extremity assistance was provided. These findings suggest that learned motor control strategies may influence movement patterns post–hip fracture

Lead Exposure Inhibits Fracture Healing and Is Associated with Increased Chondrogenesis, Delay in Cartilage Mineralization, and a Decrease in Osteoprogenitor Frequency

Lead exposure continues to be a significant public health problem. In addition to acute toxicity, Pb has an extremely long half-life in bone. Individuals with past exposure develop increased blood Pb levels during periods of high bone turnover or resorption. Pb is known to affect osteoblasts, osteoclasts, and chondrocytes and has been associated with osteoporosis. However, its effects on skeletal repair have not been studied. We exposed C57/B6 mice to various concentrations of Pb acetate in their drinking water to achieve environmentally relevant blood Pb levels, measured by atomic absorption. After exposure for 6 weeks, each mouse underwent closed tibia fracture. Radiographs were followed and histologic analysis was performed at 7, 14, and 21 days. In mice exposed to low Pb concentrations, fracture healing was characterized by a delay in bridging cartilage formation, decreased collagen type II and type X expression at 7 days, a 5-fold increase in cartilage formation at day 14 associated with delayed maturation and calcification, and a persistence of cartilage at day 21. Fibrous nonunions at 21 days were prevalent in mice receiving very high Pb exposures. Pb significantly inhibited ex vivo bone nodule formation but had no effect on osteoclasts isolated from Pb-exposed animals. No significant effects on osteoclast number or activity were observed. We conclude that Pb delays fracture healing at environmentally relevant doses and induces fibrous nonunions at higher doses by inhibiting the progression of endochondral ossification

Reduced COX-2 Expression in Aged Mice Is Associated With Impaired Fracture Healing

The cellular and molecular events responsible for reduced fracture healing with aging are unknown. Cyclooxygenase 2 (COX-2), the inducible regulator of prostaglandin E2 (PGE2) synthesis, is critical for normal bone repair. A femoral fracture repair model was used in mice at either 7–9 or 52–56 wk of age, and healing was evaluated by imaging, histology, and gene expression studies. Aging was associated with a decreased rate of chondrogenesis, decreased bone formation, reduced callus vascularization, delayed remodeling, and altered expression of genes involved in repair and remodeling. COX-2 expression in young mice peaked at 5 days, coinciding with the transition of mesenchymal progenitors to cartilage and the onset of expression of early cartilage markers. In situ hybridization and immunohistochemistry showed that COX-2 is expressed primarily in early cartilage precursors that co-express col-2. COX-2 expression was reduced by 75% and 65% in fractures from aged mice compared with young mice on days 5 and 7, respectively. Local administration of an EP4 agonist to the fracture repair site in aged mice enhanced the rate of chondrogenesis and bone formation to levels observed in young mice, suggesting that the expression of COX-2 during the early inflammatory phase of repair regulates critical subsequent events including chondrogenesis, bone formation, and remodeling. The findings suggest that COX-2/EP4 agonists may compensate for deficient molecular signals that result in the reduced fracture healing associated with aging

Lead Inhibits Skeletal Wnt Signaling and Manifests in Osteoporosis

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Environmental Medicine & Toxicology, 2013.Purpose Exposure to lead (Pb) from environmental sources remains an overlooked, but serious public health risk. We believe that intoxication from Pb, starting during childhood, results in decreased attainment of peak bone mass and disruption of bone homeostasis, and thus disposes an individual to osteoporosis. Elucidating the effect of various doses and duration of Pb exposure and subsequent mechanisms of Pb on bone processes (formation, resorption) is therefore critical for understanding risk of bonerelated diseases. We may have found a novel target of Pb toxicity in osteoblasts, the cells responsible for bone formation. We hypothesize that suppression of Wnt signaling in osteoblasts by sclerostin (Scl) is a major component to the Pb-induced decrease in bone mass. Methods We used rodent models to mimic plausible Pb exposures and determine its effect on the growing and adult skeleton. First, we used a model of low-level Pb exposure in Long-Evans rats over a lifespan of 18 months. Second, we used low, moderate, and high Pb exposure in C57BL/6 mice and determined bone quality over 13 months. Lastly, we used a mouse devoid of the sclerostin gene (SOST-/-), treated them with Pb, and conducted tests to measure bone quantity, bone quality, and bone strength. Histological assessment was performed to delineate the effect of Pb on various bone processes (formation, resorption, or fatty marrow accrual) and changes in signaling pathway components. We then used Wnt pathway reporter mice (TOPgal) to assess β-catenin signaling in the context of Pb using a luminescent substrate and X-gal staining of the β- galactosidase protein. Next, we injected mice with PTH to induce ectopic bone formation in the calvaria to assay the influence of Pb on new bone growth. Finally we used various cell models to elucidate the direct effect of Pb on osteoblastogenesis, adipogenesis, and osteoclast activity. We did this using Western blotting, quantitative PCR, cellular staining, and luciferase reporter assays. Results Using DXA and micro-CT, we found that low-level Pb decreased bone mass in rats and mice. It also resulted in bones that were more susceptible to tensile and compression forces. Pb decreased osteoblastic cell numbers and osteoblast activity with no change in osteoclasts, with an accompanying elevation in Scl protein levels in the skeleton, and correspondingly reduced levels of β-catenin and Runx2 in stromal precursor cells. Pb also increased expression of peroxisome proliferator-activated receptor-γ (PPAR-γ). These findings were substantiated in vitro, where Pb directly inhibited bone nodule formation and enhanced lipid formation. High-level Pb, however, had the opposite phenotype, increased bone mass that was more resistant to flexure testing. Osteoclast numbers were increased, however, osteoclast activity was significantly reduced. In vitro, high-level Pb osteoclast activity was decreased when added in the media or within bone wafers. We found that depression of Wnt signaling was associated with increased Scl levels in vitro and in vivo. Activation of β-catenin downstream showed amelioration of Pb-inhibition in signaling activity in the TOPgal mouse. Ultimately, removal of the sclerostin gene did not prevent against Pb-induced trabecular bone loss in long bones, but did in vertebral bone. Bone strength was ameliorated with increased Wnt signaling. Bone marrow cells from the KO mouse showed improved bone formation potential and colony formation under exposure of Pb. Conclusions Low-level Pb decreased bone mass, which was found most significant during the first 3 months of life. Pb was able to disrupt mesenchymal cell signaling, by inhibiting bone formation and promoting adipogenesis, which can be explained by depression of the Wnt/β-catenin pathway. These findings also indicate that Pb inhibition of Wnt signaling is upstream of β-catenin activation. The increased bone mass caused by gain-of-function Wnt signaling (SOST-/-) was partially blunted by Pb exposure. This reveals that SOST may not be the only target of Pb effects on the Wnt signaling pathway, yet removal of SOST did improve the Pb-induced skeletal deficits in bone quality. In all, these findings may provide a molecular basis for novel therapeutic strategies to combat Pb-induced bone pathologies

The Role of Protease Activated Receptors in Osteoblast Differentiation

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pathology and Laboratory Medicine, 2010.Protease activated receptors (PARs) are G-protein coupled receptors (GPCRs) that are activated by serine proteases and initiate a variety of cellular responses including proliferation, migration, differentiation, and mitogenesis. In vivo, PARs have been found to be predominately involved with tissue repair and wound healing. Two protease activated receptors, PAR-1 and PAR-2 have been shown to be expressed on the cell membranes of bone making cells, osteoblasts. Little is known regarding their role in osteoblast maturation and bone remodeling overall. In this thesis, our efforts have addressed the goal of discovering how PARs affect osteoblast differentiation and maturation and what function PARs play in vivo in bone development. Our research aims to examine the temporal expression of the receptors during osteoblast maturation, decipher the G-protein signaling pathways involved in osteoblasts, and study the functional response of the receptors in bone remodeling environments. Localization of PAR-1 and PAR-2 expression in active sites of bone formation in addition to temporal expression of both PAR-1 and PAR-2 throughout the development and growth states of the maturing osteoblasts was examined in this work. Using specific G-protein coupled receptor (GPCR) signaling activation, we were able to demonstrate PAR-1 and PAR-2 are active during osteoblast maturation. By examining the functional responses of PAR activation in osteoblasts, we were able to connect temporal PAR expression to osteoblast differentiation and maturation processes. It is known in the literature that PARs signal through different G-protein signaling pathways based on the cell or tissue in which they are active. The G-protein signaling mechanisms by which activated PARs on osteoblast use have not been completely characterized and were therefore examined in this thesis work using inhibitor/reporter and calcium signaling assays. Using luciferase reporters specific for several GPCR signaling endpoints, including serum response element (SRE) and Nuclear factor of activated T-cells (NFAT), we were able to identify specific target pathways that PARs may be signaling through in osteoblasts. Understanding the signaling pathways in which PAR-1 and PAR-2 may signal through in bone can help us to better understand the roles in which PARs play in osteoblast expression and function. In vivo expression and function of both PAR-1 and PAR-2 were examined using PAR-1-/- and PAR-2-/- mice. Characterization of mice absent of the receptor gave insight into the function of PARs in vivo in the musculoskeletal system. Knockout mice for both PAR-1 and PAR-2, when compared to wildtype littermates, showed a significant decrease in long bone trabecular density and decreased maturation of osteoblasts grown in culture. Injections of synthetic peptide agonists for PAR-1 and PAR-2 over the calvaria showed increase in bone deposition in wildtype mice, suggesting that PARs play an important role in bone remodeling and growth. In summary, this work shows how PAR-1 and PAR-2 are important in osteoblast maturation and function. Expression of PARs on the surface of osteoblasts and the active signaling mechanisms that these receptors signal through suggest a primary role in osteoblast function. Functional studies, both in vitro and in vivo have shown evidence that PARs may play an important role in key musculoskeletal processes, including normal and abnormal remodeling of the bone surface, wound repair within the bone environment, and several inflammatory bone diseases including osteoarthritis. The simplified amino-peptide signaling mechanism in which these receptors are activated may present many therapeutic and pharmaceutical opportunities to target several known osteoblast-related bone diseases, including osteoporosis

Duality of TRIP-1 Function in Regulating Osteoblast Activity In Vitro

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pathology, 2012.The balance in the coupling between osteoclastic bone resorption and osteoblastic bone formation is necessary to keep bone mass stable throughout life. Osteoclasts release proteins during bone resorption that remain in the resorbed pit, some of which are thought to serve as attractants and activators of osteoblasts for bone formation. One of these proteins, tartrate resistant acid phosphatase (TRAP), has recently been found to interact with TGFβ receptor interacting protein-1 (TRIP-1), a protein in osteoblasts. Evidence suggests the TRAP-TRIP-1 interaction activates the TGFβ signaling and increases osteoblast markers. TRIP-1 interacts with the type II TGFβ receptor and is phosphorylated by it. In the TGFβ pathway, TRIP-1 has been implicated as a regulator of gene expression. TRIP-1 is also involved in the initiation of protein translation; suggesting it has a dual role in the cell. We hypothesized that in osteoblasts, TRIP-1 positively regulates TGFβ signaling and activates osteoblast function during bone formation. Additionally, the function of TRIP-1 may be determined by its phosphorylation state. To explore the role of TRIP-1 in osteoblasts a characterization of its expression was performed. Results demonstrated TRIP-1 was abundantly expressed in osteoblasts, and upregulated early during differentiation. Hormones and growth factors important in bone formation regulated TRIP-1 expression. Additionally, TRIP-1 loss-of-function experiments in osteoblasts led to compromised differentiation and proliferation, cell cycle arrest and decreased activation of the TGFβ and BMP signaling pathways. TRIP-1 characterization was followed with an investigation on the mechanism for the regulation of its phosphorylation and its dual role in the cell. Our findings demonstrated that TGFβ1 regulates TRIP-1 phosphorylation and nuclear translocation in a mechanism involving Serine/Threonine phosphatases. Cell fractionation revealed phospho-TRIP-1 was most exclusively present in the cytoplasm of osteoblasts. Therefore, we believe that cytoplasmic phospho-TRIP-1 is involved in protein translation and upon dephosphorylation it can translocate into the nucleus to regulate gene transcription, in a TGFβ1-regulated mechanism. In conclusion, our findings present evidence that TRIP-1 is an important protein in osteoblasts, essential for proliferation and differentiation. This work elucidated a potential role for TGFβ-mediated regulation of TRIP-1 phosphorylation state, which may regulate its dual function in the cell