Suicide gene approach using a dual-expression lentiviral vector to enhance the safety of ex vivo gene therapy for bone repair.

'Ex vivo' gene therapy using viral vectors to overexpress BMP-2 is shown to heal critical-sized bone defects in experimental animals. To increase its safety, we constructed a dual-expression lentiviral vector to overexpress BMP-2 or luciferase and an HSV1-tk analog, Δtk (LV-Δtk-T2A-BMP-2/Luc). We hypothesized that administering ganciclovir (GCV) will eliminate the transduced cells at the site of implantation. The vector-induced expression of BMP-2 and luciferase in a mouse stromal cell line (W-20-17 cells) and mouse bone marrow cells (MBMCs) was reduced by 50% compared with the single-gene vector. W-20-17 cells were more sensitive to GCV compared with MBMCs (90-95% cell death at 12 days with GCV at 1 µg ml(-1) in MBMCs vs 90-95% cell death at 5 days by 0.1 µg ml(-1) of GCV in W-20-17 cells). Implantation of LV-Δtk-T2A-BMP-2 transduced MBMCs healed a 2 mm femoral defect at 4 weeks. Early GCV treatment (days 0-14) postoperatively blocked bone formation confirming a biologic response. Delayed GCV treatment starting at day 14 for 2 or 4 weeks reduced the luciferase signal from LV-Δtk-T2A-Luc-transduced MBMCs, but the signal was not completely eliminated. These data suggest that this suicide gene strategy has potential for clinical use in the future, but will need to be optimized for increased efficiency.

Preoperative bisphosphonate treatment may adversely affect the outcome after shoulder arthroplasty.

AIMS: The aim of this study was to investigate the effects of preoperative bisphosphonate treatment on the intra- and postoperative outcomes of arthroplasty of the shoulder. The hypothesis was that previous bisphosphonate treatment would adversely affect both intra- and postoperative outcomes. PATIENTS AND METHODS: A retrospective cohort study was conducted involving patients undergoing arthroplasty of the shoulder, at a single institution. Two patients with no previous bisphosphonate treatment were matched to each patient who had received this treatment preoperatively by gender, age, race, ethnicity, body mass index (BMI), and type of arthroplasty. Previous bisphosphonate treatment was defined as treatment occurring during the three-year period before the arthroplasty. The primary outcome measure was the incidence of intraoperative complications and those occurring at one and two years postoperatively. A total of 87 patients were included: 29 in the bisphosphonates-exposed (BP+) group and 58 in the non-exposed (BP-) group. In the BP+ group, there were 26 female and three male patients, with a mean age of 71.4 years (51 to 87). In the BP- group, there were 52 female and six male patients, with a mean age of 72.1 years (53 to 88). RESULTS: Previous treatment with bisphosphonates was positively associated with intraoperative complications (fracture; odds ratio (OR) 39.40, 95% confidence interval (CI) 2.42 to 6305.70) and one-year postoperative complications (OR 7.83, 95% CI 1.11 to 128.82), but did not achieve statistical significance for complications two years postoperatively (OR 3.45, 95% CI 0.65 to 25.28). The power was 63% for complications at one year. CONCLUSION: Patients who are treated with bisphosphonates during the three-year period before shoulder arthroplasty have a greater risk of intraoperative and one-year postoperative complications compared with those without this previous treatment.

Lentiviral-mediated BMP-2 gene transfer enhances healing of segmental femoral defects in rats.

The objective of the present study was to assess the ability of bone marrow cells expressing BMP-2 created via lentiviral gene transfer to heal a critical sized femoral defect in a rat model. Femoral defects in Lewis rats were implanted with 5x10(6) rat bone marrow stromal cells (RBMSC) transduced with a lentiviral vector containing either the BMP-2 gene (Group I), the enhanced green fluorescent protein (LV-GFP) gene (Group IV), or RBMSC alone (Group V). We also included femoral defects that were treated with BMP-2-producing RBMSC transduced with lentivirus, 8 weeks after infection (Group III), and a group with 1x10(6) RBMSC transduced with a lentiviral vector with the BMP-2 gene (Group II). All defects (10/10) treated in Group I healed at 8 weeks compared with none of the femora in the control groups (Groups IV and V). In Group II, only one out of 10 femora healed. In Group III, 5 out of 10 femora healed. Significantly higher amounts of in vitro BMP-2 protein production were detected in Groups I, II, and III when compared to that of the control groups (p<0.05). Histomorphometric analysis revealed significantly greater total bone volume in defects in Group I and III when compared to control specimens (p<0.003). Biomechanical testing revealed no significant differences in the healed defects in Groups I and III when compared to intact, nonoperated femora with respect to peak torque and torque to failure. Our results indicate that BMP-2-producing RBMSC created through lentiviral gene transfer have the capability of inducing long-term protein production in vitro and producing substantial new bone formation in vivo.

Effects of phenol and 2,6-dimethoxyphenol (syringol) on in vivo formation of N-nitrosomorpholine in rats.

We determined the effects of phenol and 2,6-dimethoxyphenol (syringol) on N-nitrosomorpholine (NMOR) formation in rats given morpholine and nitrite by gavage. At 30 min post-gavage the recovery (from the stomach, duodenum and blood) of 564 micrograms NMOR was six times higher when administered to rats by gavage with 2 g of semipurified diet (SPD) than when given without food. Rats were gavaged with 12 mg each of morpholine, one of the modifiers and nitrite and examined 30 min later. Syringol decreased the amount of NMOR in both the stomach and blood by 89%, while phenol had no effect. We compared these results with those obtained with ascorbic acid and thiocyanate. The effect of ascorbic acid was similar to that of syringol. However, thiocyanate increased the amount of NMOR in the stomach and blood 2.7- and 4-fold, respectively. When 2 g of SPD was administered to rats by gavage, together with the precursors, syringol and ascorbic acid blocked NMOR formation in the stomach by 58 and 45%, respectively, and thiocyanate enhanced the yield 1.5-fold. The effect of phenol was not significant for the stomach and blood and that of the other modifiers was not significant for blood. Administration of the reactants together with food decreased the NMOR level in blood 155-fold relative to controls (no food), suggesting that food decreased the absorption rate over a 30-min period. These results demonstrate the modifying effect of phenol and syringol on NMOR formation in vivo to be similar to that observed in a previous in vitro study, and show that the effect of food on NMOR levels in blood was more important than that of the modifiers.