The role of transduced bone marrow cells overexpressing BMP-2 in healing critical-sized defects in a mouse femur.

The role that transduced mouse bone marrow stromal cells (mBMSCs) engineered to overexpress human bone morphogenetic protein 2 (BMP-2) play in healing critical-sized skeletal defects is largely unknown. We evaluated the interaction between host osteoprogenitor cells and donor mBMSCs transduced with either a lentiviral (LV) vector-expressing red fluorescent protein (RFP) with or without BMP-2 that were implanted into a critical-sized femoral defect. Radiographs taken at the time of killing were evaluated using a five-point scaled scoring system. Frozen histologic sections were analyzed to assess both the transduced cells' role in bone repair and the local osteoprogenitor response. There was complete radiographic bridging in 94% of group I (LV-RFPch-BMP-2-cmyc) and 100% of group III (recombinant human BMP-2) specimens. Radiographs demonstrated a lack of healing in group II (LV-RFPch). Mouse BMSCs transduced with an LV-RFPch-BMP-2 vector were able to induce host cells to differentiate down an osteoblastic lineage and heal a critical-sized defect. However, the donor cells appeared to be functioning as a delivery vehicle of BMP-2 rather than actually differentiating into osteoblasts capable of participating in bone repair as evidenced by a lack of colocalization of the transduced cells to the sites of skeletal repair where the host progenitor cells were found.

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.

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.

[Perinatal and postnatal study of cefpirome sulfate in rats].

A perinatal and postnatal study was performed in rats by intraperitoneal administration of cefpirome sulfate (CPR) at dose levels of 0 (control), 200, 400 and 800 mg/kg/day to dams from day 17 of pregnancy to day 21 after delivery. Twenty one or twenty two dams in each group were allowed to deliver for the postnatal examination of their offsprings. No animal died. Loose feces were observed in the 800 mg/kg/day group. Body weight gain of the dams was retarded in the 800 mg/kg/day group in the early stage of treatment. Food intake was reduced simultaneously in the 200, 400 and 800 mg/kg/day groups. Autopsy revealed the enlargement of caecum in the 800 mg/kg/day. The adrenal weight was increased in the 400 and 800 mg/kg/day groups, and the kidney weight was increased in the 800 mg/kg/day group. The body weight of pups at birth in the 800 mg/kg/day group was slightly lower than that in the control group. The body weight of male pups in the 800 mg/kg/day group decreased transiently during rearing period. However, CPR had no adverse effects on the postnatal development such as viability, differentiation, emotionality, learning ability or reproductive performance. The results suggest that the non-effective dose level of CPR is lower than 200 mg/kg/day for general toxicity, 800 mg/kg/day for reproductive ability in maternal animals and 400 mg/kg/day in offsprings respectively.

[Fertility study of cefpirome sulfate in rats].

A fertility study was performed in rats by intraperitoneal administration of cefpirome sulfate (CPR) at dose levels of 0 (control), 200, 400 and 800 mg/kg/day. Male rats were treated for 60 days before mating and during the mating period. Female rats were administrated the substance from 14 days before mating to day 7 of pregnancy. The females were sacrificed on day 21 of pregnancy for examination of their fetuses. No animal died during the administration period. Loose feces were observed in both male and female animals in the 400 and 800 mg/kg/day groups. Body weight gain was suppressed in both male and female animals in the 800 mg/kg/day group and in male animals in the 400 mg/kg/day group. There were no significant differences in food and water intakes between treated and control groups. Autopsy revealed the enlargement of the caecum in males in the 200, 400 and 800 mg/kg/day groups. The kidney and adrenal weights were significantly increased in males in the 200, 400 and 800 mg/kg/day groups. Fertility and reproductive ability in both sexes, and estrus cycles in female rats were not affected by administration of CPR. There were no lethal effect and growth-inhibiting or teratogenic effects on the embryos and the fetuses. The results suggest that the non-effective dose level of CPR was lower than 200 and 200 mg/kg/day for general toxicity in male and female parent animals respectively, 800 mg/kg/day for reproductive ability in parent animals and in embryos and fetuses.

[Teratological study of cefpirome sulfate in rats].

A teratogenicity study was performed in rats by intraperitoneal or intravenous administration of cefpirome sulfate (CPR) at dose level of 0 (control), 200, 400 and 800 mg/kg/day to dams from day 7 to day 17 of pregnancy. Twenty one to twenty eight female rats in each intraperitoneal and intravenous administrated group were sacrificed on day 21 of pregnancy for examination of their fetuses, and ten to thirteen female rats in each intraperitoneal administrated group were allowed to deliver for the postnatal examination of their offsprings. In the 800 mg/kg/day intravenous administrated group, two dams out of twenty four died during administration period, however no animal died in any intraperitoneal administrated group. The doses of 400 and 800 mg/kg/day caused piloerection, diarrhea or loose feces in the both administration routes, and accelerated breathing, a decrease in spontaneous activity, systemic spasms, cataleptic symptoms and wild running in the intravenous route. The suppression of body weight gain was detected in the 800 mg/kg/day intraperitoneal administrated group and 200, 400 and 800 mg/kg/day intravenous administrated groups, however there were no significant differences in food and water intakes between treated and control groups. At autopsy, enlargement of caecum and an increase in adrenal weight were detected in the 800 mg/kg/day groups of both administration routes. Body weight of the fetuses was decreased in both sexes in the 800 mg/kg/day group and in male fetuses in the 400 mg/kg/day group, and placental weight was decreased in the 800 mg/kg/day group, in the both administration routes respectively. However, embryonal or fetal mortality and incidences of external or visceral anomalies were not increased. In offspring, the dose of 800 mg/kg/day caused very slight suppression of body weight gain, however CPR had no adverse effects on the postnatal development such as viability, differentiation, emotionality, learning ability or reproductive performance. The results suggest that the non-effective dose level of CPR is 200 mg/kg/day in maternal animals and fetuses, 400 mg/kg/day in offsprings in intraperitoneal route, lower than 200 mg/kg/day in maternal animals and 200 mg/kg/day in fetuses in intravenous route respectively.

[The effect of the age factor on open-field behavior in rats].

We examined the age-related behavior under open-field conditions using 925 Wistar-Imamichi rats of both sex ranging 21 to 695 days old. The mean score of ambulation and rearing increased between 28-56 days old, and both decreased after those ages. The coefficients of variation were stable and kept minimum values between 28-98 days old. Other parameters, except for ambulation and rearing, did not show the age-related change. Each parameter was divided into 3 one-minute-values in order to detect the change in minutes after exposure in open-field conditions. The chronological change in each behavioral parameter has its own specific pattern regardless of age and sex. Namely, it has been proved that a rat has an invariable natural behavioral pattern with regard to a time course in open-field conditions. It is important to detect a change of chronological pattern in each parameter, because an abnormal subject would be identified by an appearance of change in the chronological pattern. Sexual difference also was identified in all ages. It is necessary to evaluate separately parameters depending on sexual difference.

[An application of in vitro fertilization--embryo culture--embryo transfer system on the drug safety evaluation: fertility test of male mice administered with the anticancer drug].

We examined an application of in vitro fertilization--embryo culture--embryo transfer system for reproductive and developmental study on the drug safety evaluation in mice. The male mice at 10 weeks of age were administered intravenously with a single dose of 75 mg/kg of the anticancer platinum complex (DWA 2114R) which inhibits DNA synthesis. Four to six weeks after administration, the males were mated with the superovulated females. Fertilization rates were significantly lower than the controls at each weeks after the administration. Furthermore, delayed formation of pronucleus was observed as compared with the control. Four weeks after administration, the preimplantation development to blastocyst stage of those embryos in vitro and the survival rates on the day 17 of gestation after embryo transfer suggested that a DNA synthesis in germ cells during maturation was inhibited and/or prevented by DWA 2114R. The results of in vitro fertilization reflected its sperm concentration rather than the administration of DWA 2114R. Thus, an analysis of the delayed formation of pronucleus observed fertilization in vivo could not done in detail. To use for the drug safety evaluation, there exist plenty of room for improvement in this system. These results have showed that the embryo culture and the embryo transfer are useful techniques as the reproductive and developmental study on the drug safety evaluation. These techniques bring additional informations on the pre- and post-implantation development in vivo.