Edward J. Puzas

Evidence for a direct role of cyclo-oxygenase 2 in implant wear debris-induced osteolysis.

Aseptic loosening is a major complication of prosthetic joint surgery and is manifested as chronic inflammation, pain, and osteolysis at the bone implant interface. The osteolysis is believed to be driven by a host inflammatory response to wear debris generated from the implant. In our current study, we use a selective inhibitor (celecoxib) of cyclo‐oxygenase 2 (COX‐2) and mice that lack either COX‐1 (COX‐1−/−) or COX‐2 (COX‐2−/−) to show that COX‐2, but not COX‐1, plays an important role in wear debris‐induced osteolysis. Titanium (Ti) wear debris was implanted surgically onto the calvaria of the mice. An intense inflammatory reaction and extensive bone resorption, which closely resembles that observed in patients with aseptic loosening, developed within 10 days of implantation in wild‐type and COX‐1−/− mice. COX‐2 and prostaglandin E2 (PGE2) production increased in the calvaria and inflammatory tissue overlying it after Ti implantation. Celecoxib (25 mg/kg per day) significantly reduced the inflammation, the local PGE2 production, and osteolysis. In comparison with wild‐type and COX‐1−/− mice, COX‐2−/− mice implanted with Ti had a significantly reduced calvarial bone resorption response, independent of the inflammatory response, and significantly fewer osteoclasts were formed from cultures of their bone marrow cells. These results provide direct evidence that COX‐2 is an important mediator of wear debris‐induced osteolysis and suggests that COX‐2 inhibitors are potential therapeutic agents for the prevention of wear debris‐induced osteolysis.

Malignant reversion of a human osteosarcoma cell line, Saos-2, by inhibition of NFκB

Beyond a pivotal role in neoplastic transformation and malignant progression, NFkappaB is intricately involved in bone biology, pointed up by the osteopetrotic phenotype of NFkappaB (p50-p52) double knock-out mice. Osteopetrosis results from intrinsic defects in osteoclastogenesis, loss of osteoclast bone resorptive activity and, questionably, increased osteoblast activity (bone matrix apposition and mineralization). We here report that inhibition of NFkappaB signaling activity in Saos-2 cells results in a marked decrease in cellular proliferation, assessed by the incorporation of radioactive thymidine into cellular DNA. Decreased cellular proliferation was accompanied by the induction of bone morphogenic proteins (BMP) 4, 7, and the osteoblast specific transciption factor, Cbfa1, heralding osteoblast differentiation, given the induction of alkaline phosphatase, osteopontin, and osteocalcin message levels and the attendant increase in matrix deposition and mineralization in vitro. These results point to the negative regulation of osteoblast differentiation by NFkappaB, with implications in the pathogenesis and progression of osteosarcomas.

Vertebral body osteopenia associated with posterolateral spine fusion in humans

Study Design Lateral dual‐energy x‐ray absorptiometry was used to examine isolated changes in vertebral body mineral density in humans after instrumented posterolateral lumbar fusion. Objectives To determine if device‐related osteopenia will occur in humans who undergo spinal fusion. Device‐related osteopenia is known to occur as a result of local stress shielding after instrumentation in the appendicular skeleton. This effect has not been observed, however, in humans after spine fusion. To evaluate such changes, the vertebral body mineral density was measured in eight patients who had instrumented lumbar fusion and in eight matched control patients who had lumbar surgery with no fusion. Summary of Background Data In previous studies of dogs, vertebral body osteopenia occurred as a result of instrumented spine fusions. Previous studies in humans, however, have been limited by the relative insensitivity of conventional photon absorptiometry to isolated changes in the vertebral body because of overlying posterior elements. Methods Absorptiometry was performed an average of 31.9 months after posterolateral fusion that bridged at least one level in the region of L2‐L4. To reduce the effects of individual variations in mineral metabolism, the vertebral values were standardized by using the ratio of vertebral body to femoral neck density for each patient. Results The mean density ratio for the group of patients who underwent spine fusion was 0.733. This value was significantly lower than the control ratio of 0.879 (P = 0.048). Conclusions Patients who have undergone instrumented posterolateral lumbar fusions have decreased vertebral body bone mineral density at the level of fusion compared with that of matched controls.

The Mevalonate Synthesis Pathway as a Therapeutic Target in Cancer

Farnesyl transferase inhibitors have emerged as bona fide anticancer agents whereas the development of geranylgeranyl transferase inhibitors has been mitigated by overt systemic toxicities. Evidence suggests that the therapeutic value of farnesyl transferase inhibitors is an indirect result of perturbations in the function of geranylgeranylated Rho proteins. To address this question, we used inhibitors of the mevalonate synthesis pathway to decrease cellular levels of farnesly and geranylgeranly isoprenoids and supplemented our culture systems with exogenous isoprenoids accordingly. Using a murine lung alveolar carcinoma cell line (Line 1), we report a dose-dependent inhibition of tumor cell proliferation, adhesion and invasiveness, in response to alendronate (3–30 μmol/L) and mevastatin (1–10 μmol/L). Supplementation of cultures with geranylgeranyl pyrophosphates (100 μmol/L) was observed to rescue drug-induced phenotypic changes whereas farnesyl pyrophosphate (100 μmol/L) had a minimal effect. Our observations highlight the mevalonate synthesis pathway as a target for anticancer therapies and suggest a greater role for geranylgeranylated proteins in cellular processes germane to cancer.

Synergism of aminobisphosphonates and farnesyl transferase inhibitors on tumor metastasis

Aminobisphosphonates have shown significant antitumor activity in vitro and in vivo with selective pharmacodistribution to bone, and an established role in the treatment of malignant bone disease. Given that the mode of action of aminobisphosphonates involves decreasing the prenylation of the Rho family of proteins, through decreasing the availability of prenyl groups (farnesyl and geranylgeranyl isoprenoids), the authors sought the inhibition of Rho protein prenylation at two points, by using an aminobiphosphonate (alendronate) in conjunction with a prenyl transferase inhibitor (R115777, a specific farnesyl transferase inhibitor with limited effects in geranylgeranyl transferase). The authors show synergistic inhibition of the prenylation dependent membrane association and migratory function of Rho proteins, translating into a suppressive effect on in vitro tumor cell invasiveness and in vivo metastasis. The findings support the use of aminobisphosphonates in conjunction with farnesyl transferase inhibitors in the prevention of metastatic progression and suggest that metastatic progression is a valid end point in assessing the antitumor activity of farnesyl transferase inhibitors.

Facial bone density: effects of aging and impact on facial rejuvenation.

BACKGROUND Facial bone aging has recently been described as primarily resulting from volume loss and morphologic changes to the orbit, midface, and mandible. OBJECTIVE The authors demonstrate how the facial skeleton bone mineral density (BMD) changes with age in both men and women and compare these changes to those of the axial skeleton. They also explore the aesthetic implications of such changes in bone density. METHODS Dual-energy X-ray absorptiometry (DXA) scans of the facial bones and lumbar spine were obtained from 60 white subjects, 30 women and 30 men. There were 10 men and 10 women in each of 3 age categories: young (20-40 years), middle (41-60 years), and old (61+ years). The following measurements were obtained: lumbar spine BMD (average BMD of L1-L4 vertebrae), maxilla BMD (the average BMD of the right and left maxilla), and mandible BMD (the average BMD of the right and left mandibular ramus). RESULTS The lumbar spine BMD decreased significantly for both sexes between the middle and old age groups. There was a significant decrease in the maxilla and mandible BMD for both sexes between the young and middle age groups. CONCLUSIONS Our results suggest that the BMD of the face changes with age, similar to the axial skeleton. This change in BMD may contribute to the appearance of the aging face and potentially affect facial rejuvenation procedures.

Clinical relevance of increased retinoid and cAMP transcriptional programs in tumor cells rendered non-malignant by dominant negative inhibition of NFκB

We previously reported reciprocal regulation of extracellular matrix degrading enzymes and their endogenous inhibitors by NFkappaB. As such, dominant negative inhibition of NFkappaB in a murine lung alveolar carcinoma cell, Line 1, afforded a decrease in malignant proclivity [Cancer Res. 60(23) (2000) 6557-6562]. Contrasting the gene expression profile of malignant Line 1 tumor cells (WT-Line 1) and their non-malignant counterparts transduced with a dominant negative inhibitor of NFkappaB (mIkappaB-Line 1), we observed upregulated retinoic acid receptors (RARs) and the cAMP response element modulator (CREM), in mIkappaB-Line 1 tumor cells, and utilized heterologous promoter-reporter constructs to confirm enhanced responsiveness. We translate these findings by inducing retinoid and cAMP transcriptional programs in WT-Line 1 tumor cells with pharmacologic doses of all-trans retinoic acid (at-RA) and pentoxyfilline (PTX), respectively, and demonstrate suppression of NFkappaB activity, tumor cell derived matrix metalloprotease 9 activity, tumor cell invasiveness in vitro and spontaneous metastasis in vivo. Our results are consistent with the putative role of retinoids and cAMP in the malignant reversion of tumor cells and illustrative of the binary nature of transcriptional programs that modulate malignant progression.

A genome-wide expression profile and system-level integration of nuclear factor kappa B regulated genes reveals fundamental metabolic adaptations during cell growth and survival

A murine lung alveolar carcinoma cell line (WT‐Line 1) and its equally tumorigenic but non‐malignant derivative transduced with a dominant negative inhibitor of NF‐κB (mI‐κB‐Line 1), were profiled on the Affymetrix® 19 000 gene array platform. Two differentially expressed gene clusters were identified and integrated into a functional model. The downregulation of anti‐oxidant defenses, in mI‐κB‐Line 1 cells, correlates with high levels of reactive oxygen species (ROS) and ROS damage to cellular macromolecules while the upregulation of metabolic nuclear receptors correlates with an adaptive/survival response, which involves a shift in energy metabolism toward β‐oxidative respiration. Accordingly, mI‐κB‐Line 1 cells are markedly sensitized to pharmacologic inhibition of β‐oxidative respiration. These findings are indicative of compensatory changes that could undermine anti‐cancer therapies targeting NF‐κB.

Allogeneic and Autogenous Bone Grafts Are Affected by Historical Donor Environmental Exposure

BackgroundBone graft materials are routinely evaluated for infectious agents; however, data regarding contamination of bone graft from environmental exposure of the donors to osteotoxic substances such as lead are not routinely available. In animal models, stored lead in bone has been shown to impair fracture healing and osteocyte function. In clinical studies, lead is linked to skeletal disease at relatively low concentrations. Presumably the levels of lead in allografts mirror the level of lead in bone in the population; however, the degree to which processing might decrease this and the frequency with which potentially osteotoxic levels appear in bone grafts have not been studied.Questions/purposes(1) Does processing of donor bone for allografts result in lower concentrations of lead in commercial allograft when compared with autologous bone graft; and (2) what proportion of bone grafts contain potentially osteotoxic levels of lead from > 2.0 to 20.0 µg/g corresponding to environmental exposure?MethodsAllograft from commercial sources and autologous bone graft materials were examined for lead content using ICP- atomic absorption spectrophotometric analysis. We analyzed bone graft specimens from 42 donors, including 26 corticocancellous tibial specimens from commercially available bone graft materials and 16 autograft corticocancellous tibial specimens. Lead levels were determined for the cortical (n = 42) and cancellous (n = 42) portions of each specimen. For quality control, all instruments, plastic and glassware, were regularly tested for lead contamination by atomic absorption spectrophotometry throughout the experiments. In addition, spectrophotometer calibration was verified using Standard Reference Material 1486 bone meal (NIST, Gaithersburg, MD, USA). Descriptive statistical analysis was performed using SPSS 20 (SPSS Inc, Chicago, IL, USA). Using these techniques, a lead level > 2 µg/g to 20 µg/g corresponds to some degree of environmental exposure to lead.ResultsWith the numbers available in the present study, there were no differences in mean lead level between commercial bone graft materials and autogenous bone graft, 2.1 µg/g (95% confidence interval [CI], 1.6–3.3 µg/g) versus 2.0 µg/g (95% CI, 1.0–4.5 µg/g; p = 0.86). The range for all tested samples varied from < 0.1 to 5.0 µg/g. Likewise, there were no differences in mean lead level between cortical bone grafts, which contained 2.2 µg/g (95% CI, 1.5–3.7 µg/g), and cancellous grafts, which contained 1.9 µg/g (95% CI, 1.2–3.4 µg/g; p = 0.58). Thirty-eight percent (16 of 42) of the specimens had levels between 2.0 µg/g and 20 µg/g within a range expected for individuals with known environmental exposure to lead.ConclusionsThis study demonstrates that lead is present in up to one-third of tibial allograft and autograft bone specimens at potentially osteotoxic levels regardless of the source or screening. Further research is needed to delineate the relationship with nonunion or pseudoarthrosis after procedures in which allograft is used. In addition, further study would examine concentrations of lead and other environmental contaminants in other graft types.Clinical RelevanceComparable levels of lead exposure have been associated with toxic effects on skeletal tissue. Further study of bone graft used in fusion procedures and other procedures is necessary to define the magnitude of osteotoxic effects in the setting of fracture care or fusion procedures.