To evaluate the roles or effects of oviductus ranae (OR) or oviductus ranae eggs (ORE) in preventing and treating postmenopausal osteoporosis.

In vivo experiment: Sixty female adult Wistar rats were randomly divided into 5 groups of 12. To provide an osteoporosis model 4 groups of rats were ovariectomized (OVX), with the 5th being sham operated. Medication commenced 7 days after the operation and lasted continuously for 12 weeks. Sham operated and OVX groups were given equivalent volumes of 5% Tween-80. The other three groups intragastrically received conjugated estrogens (CE), OR or ORE of the corresponding doses. At the 12th week, serum estrogen, bone gla protein (BGP), serum calcium, phosphorus, and alkaline phosphatase (ALP) were assayed; bone mineral densities (BMD) were measured and bone scanning was conducted; uteri were weighed, and weight, volume and length of the femoral bones were determined; and cortical thickness of femoral heads and area of bone trabecula were measured by image analyzer. In vitro experiment: Eighty 10-month old SD rats, with equal numbers of males and females, were randomly divided into 8 groups. Osteoblasts were isolated from neonatal rat calvariae, and the cells were exposed to various concentrations of serum from OR and ORE groups to study the impact of these sera on osteoblastic proliferation, ALP activity and mineralization. Osteoclastic numbers were determined using tartrate resistant acid phosphatase (TRAP).

In vivo experiment: The body weight of the four OVX groups increased significantly (P<0.01). Uterine weight of the CE group was the highest (P<0.01); Compared with the model group, estrogen level, BMD, bone scanning/bone imaging index weight of the femoral bones, cortical thickness of femoral heads in the OR and ORE groups increased significantly (P<0.05, P<0.01); femoral volume in the ORE group increased significantly (P<0.05); and the content of osteocalcin, phosphorus, and ALP in serum decreased significantly (P<0.05, P<0.01). In vitro experiment: Sera from OR and ORE groups had notable effects on the proliferation of osteoblasts (P<0.05 and P<0.01, repsectively) and stimulated the formation of calcium nodes (P<0.05, P<0.01), while the enhancement of ALP activity in osteoblasts was significant (P<0.05, P<0.01). The number of TRAP-positive cells was significantly reduced as well (P<0.01).

OR and its eggs could effectively suppress OVX-induced osteoporosis in rats, and increase bone turnover possibly by both an increase in osteoblastic activity and a decrease in osteoclastic activity. The present study provides evidence that OR and its eggs could be considered a complementary and alternative medicine for the treatment of postmenopausal osteoporosis.

Gene transfer into stem cells has been an ongoing priority as a treatment for genetic disease and cancer for more than two decades. Methods described herein, form the basis for providing the cell source to determine if osteoclast precursor cells (OcP) can be used as therapeutic gene delivery systems in vivo. Osteoclasts and tumor associated macrophages or OcP, support survival, tumor progression and osteolysis in bone cancers. Two sources of precursor cells are compared: CD14+ cells, the standard OcP, found abundantly in peripheral blood and CD34+ cells, hematopoietic stem cells that are rare, but which can be expanded into OcP. Our findings characterize cell yield at each step of the transduction process and thus provide essential data for planning future in vivo experiments. In addition we demonstrate that essential functions of OcP are preserved following lentiviral transduction. Specifically, neither the transduction method nor the lentiviral transduction influence the OcP's ability to form osteoclasts, express the marker gene, EGFP, or resorb bone. Finally, we conclude that CD34+ cells yield significantly more transduced cells and form functionally superior osteoclasts in vitro. This study represents a step towards considering human gene therapy for bone cancer by demonstrating successful transduction of human OcP for use as cellular delivery vehicles to sites of bone cancer.

This study evaluates the effect of alendronate on osteoclastogenesis, cytokine production, and bone resorption in postmenopausal women. We suggest that it acts on mature bone resorbing osteoclasts after 3 months of treatment, whereas, after 1 year, it diminishes their formation by reducing their precursors and serum RANKL.

Osteoclasts are the target cells of bisphosphonates, though the most drug-sensitive steps of their formation and activity have not been determined. The present study evaluates the effect of alendronate on osteoclastogenesis, cytokine production, and bone resorption in postmenopausal women.

The study was conducted on 35 osteoporotic women; 15 were pretreated with alendronate 70 mg/week, whereas, 20 were treated with calcium 1 g/day and vitamin D 800 IU/day. After 3 months, 30 received alendonate 70/mg, vitamin D 2800 IU/week, and calcium 1 g/day for 12 months (combined therapy), whereas, the other five patients remained on calcium 1 g/day and vitamin D 800 IU/day. The following parameters were assessed before and after therapy: changes in bone resorption markers, circulating osteoclast precursors, formation of osteoclasts in peripheral blood mononuclear cell cultures, their viability, and variations in cytokines production.

After 3 months of alendronate, there was no significant reduction in the number of osteoclast precursors, osteoclast formation and viability, and cytokine levels, whereas, there was a significant reduction of bone resorption markers. One year of the combined therapy, on the other hand, reduced osteoclast precursors, osteoclast formation, and serum RANKL, whereas, calcium plus vitamin D alone had no effect.

We suggest that alendronate mainly acts on mature bone resorbing osteoclasts in the short term, whereas, its long-term administration diminishes their formation by reducing their precursors and serum RANKL.

Fracture healing is a complex process that involves several cell types; as a previous report suggested an increase in osteoblast (OB) precursors in peripheral blood during this process, this paper examines the role of circulating bone cell precursors in this process in the light of a prior suggestion that OB precursors are increased. Nine healthy men less than 60 years old with traumatic fractures were enrolled. The parameters circulating OB precursors (osteocalcin+/alkaline phosphatase+/CD15- cells) and osteoclast precursors (CD14+/CD11b+/vitronectin receptor + cells) were measured by flow cytometry; bone formation markers and TGFbeta1, by ELISA; and PTH, by RIA in serum on arrival at the emergency department (baseline) and 15 days after fracture. Bone cell precursors behaved differently during healing. TGFbeta1 was inversely correlated with OB number, but increased their degree of maturation at baseline. Bone formation markers and TGFbeta1 were increased after fracture, whereas PTH was decreased. The TGFbeta1 increase was directly correlated with age, whereas age was not correlated with the precursors. In conclusion, we confirm the role of TGFbeta1 in fracture healing; and its possible role in the control of pre-OB homeostasis. There was no variation in circulating precursor cells during healing, though the increase in TGFbeta1 may suggest increased pre-OB maturation and homing to the injured site.

Compelling evidences suggest that increased production of osteoclastogenic cytokines by activated T cells plays a relevant role in the bone loss induced by estrogen deficiency in the mouse. However, little information is available on the role of T cells in post-menopausal bone loss in humans. To investigate this issue we have assessed the production of cytokines involved in osteoclastogenesis (RANKL, TNFalpha and OPG), in vitro osteoclast (OC) formation in pre and post-menopausal women, the latter with or without osteoporosis. We evaluated also OC precursors in peripheral blood and the ability of peripheral blood mononuclear cells to produce TNFalpha in both basal and stimulated condition by flow cytometry in these subjects. Our data demonstrate that estrogen deficiency enhances the production of the pro-osteoclastogenetic cytokines TNFalpha and RANKL and increases the number of circulating OC precursors. Furthermore, we show that T cells and monocytes from women with osteoporosis exhibit a higher production of TNFalpha than those from the other two groups. Our findings suggest that estrogen deficiency stimulates OC formation both by increasing the production of TNFalpha and RANKL and increasing the number of OC precursors. Women with post-menopausal osteoporosis have a higher T cell activity than healthy post-menopausal subjects; T cells thus contribute to the bone loss induced by estrogen deficiency in humans as they do in the mouse.

Gene therapy, an applied form of biotechnology, relies on the delivery of foreign DNA into cells. More than 50% of all reported clinical trials for gene therapy are for cancer, though only a scant number for osteosarcoma. Osteosarcoma is a neoplasm afflicting young adults, who in their prime years of life suffer debilitation if not death. The disease is not entirely curable, even with surgery combined with aggressive chemotherapy. Thus, other forms of therapies are being evaluated, including gene therapy. There exist two major forms of gene transfer: viral and non-viral. This review only covers proof-of-principle work carried out in cancer beyond the cell culture stage, in animals. Drawing from the experiences of gene therapy against other cancers, studies for which have already reached the clinical phase, the review discusses potential pitfalls and solutions to enhance gene therapy for osteosarcoma.

This paper studies the effect of oral risedronate on osteoclast precursors, osteoclast formation, and cytokine production in 25 osteoporotic women. Risedronate is effective in reducing the number of osteoclast precursors, their formation, vitality, and activity and the level of RANKL and TNF-alpha in cultures.

Bisphosphonates inhibit bone resorption by acting against osteoclasts. Some in vitro studies suggest that they induce osteoclast apoptosis; others suggest that they exert an effect on the production of pro-osteoclastogenic cytokines. The effect of risedronate on osteoclastogenesis by peripheral blood mononuclear cells (PBMCs) in postmenopausal osteoporosis has not been previously studied. This paper examined the influence of risedronate on the formation of osteoclast precursors and cytokine production within the compass of osteoclastogenesis in osteoporosis.

This study was conducted on 38 osteoporotic women; 25 patients were treated with risedronate 5 mg/d, whereas 13 were treated with calcium 1 g/d and vitamin D 800 UI/d. The following parameters were assessed: changes in bone turnover, circulating osteoclast precursors, formation of osteoclasts in PBMC cultures, their activity and vitality, and variations in the production of pro-osteoclastogenic cytokines before and after therapy.

After 3 mo of risedronate, there was a significant reduction in the number and degree of differentiation of osteoclast precursors, osteoclast formation, vitality and activity, and in the level of RANKL and TNF in cultures and of TNF and osteoprotegerin (OPG) in serum, whereas in the group treated with calcium and vitamin D, there were no significant changes.

Our data show that risedronate is effective in lowering the number of circulating osteoclast precursors, their formation, vitality, and activity in cultures, and in reducing the level of pro-osteoclastogenic cytokines in culture supernatants and in serum.

This review begins with an introduction to the malignant bone tumor, osteosarcoma [OS] and then moves to a discussion of the commonly used vectors for gene transfer. We first briefly highlight non-viral vectors including polymeric and liposomal delivery systems but concentrate predominantly on the 5 leading viral vectors used in cancer gene therapy, specifically retroviruses, adeno-associated viruses, herpes viruses and lentiviruses with the most detailed analysis reserved for adenoviruses. The 3 main strategies for gene therapy in osteosarcoma are next summarized. As part of this review, the several prodrug-converting enzymes utilized in OS suicide gene therapy are examined. The text then turns to a discussion of adenovirus-mediated gene transfer and the need for tumor targeting via transductional or transcriptional approaches. Because of practical problems with use of replication-incompetent viruses in achieving complete tumor kill in vivo, virotherapy utilizing replication competent viruses has come to the fore. This topic is, thus, next reviewed which allows for a natural transition to a discussion of armed therapeutic viruses many of which are conditionally replicating adenoviruses carrying transgenes with established anti-tumor efficacy. We recognize that several other issues have arisen which hamper progress in the field of cancer gene therapy. We, therefore, review viral-induced toxicity in the host and vector delivery issues which have been found to potentially influence safety. We end with a brief perspective including commenting on animal models used in examining delivery strategies for osteosarcoma gene therapy. The challenges remaining are touched upon most especially the need to deal with pulmonary metastatic disease from OS.

Primary and metastatic bone cancers are difficult to eradicate and novel approaches are needed to improve treatment and extend life. As bone cancer grows, osteoclasts, the principal bone-resorbing cells of the body, are recruited to and activated at sites of cancer. In this investigation, we determined if osteoclast lineage cells could function as a cell-based gene delivery system to bone cancers. We used the cytosine deaminase (CD) 5-fluorocytosine (5-FC) enzyme/prodrug system and studied bone marrow and bones from transgenic mice expressing a novel CD gene regulated by the osteoclast tartrate-resistant acid phosphatase (TRAP) gene promoter (Tg/NCD). DsRed2-labeled 2472 sarcoma cells were placed in Tg/NCD osteoclastogenic cultures and treated with 5-FC. 5-FC treatment resulted in profound bystander killing (90%; P < 0.05). The effect of 5-FC treatment on osteoclast lineage cells was most dramatic when administered at the beginning of the 7-day cultures, suggesting that mature osteoclasts are less sensitive to 5-FC. Evaluation of osteoclast-directed bystander killing in vivo revealed dramatic killing of bone cancer with only a modest effect on osteoclast number. Specifically, 5-FC treatment of tumor-bearing Tg/NCD mice or Tg/NCD bone marrow transplanted C3H mice (Tg/NCD-C3H) resulted in 92% and 44% reductions in tumor area, respectively (P < 0.05). Eight of ten 5-FC-treated Tg/NCD mice had complete bone tumor killing and five of six 5-FC-treated Tg/NCD-C3H mice had reduced tumor compared with controls. In addition, Tg/NCD osteoclasts were resistant to 5-FC treatment in vivo, a very important feature, as it identifies osteoclasts as an ideal CD gene delivery system.

Painful breast carcinoma metastases in bone are a common manifestation of malignant disease. Eradication of these tumors can be evasive, and as a result, skeletal morbidity increases with disease progression.

The treatment potential of cytosine deaminase (CD) gene therapy combined with radiation treatment was evaluated in vitro and in vivo using a 4T1 murine breast carcinoma model. 4T1 carcinoma cells were transduced with a fusion gene encoding the extracellular and transmembrane domains of the human nerve growth factor receptor and the cytoplasmic portion of the yeast CD gene (NGFR-CD(y)).

CD-expressing tumor cells (4TCD(y)) were highly sensitive to treatment by 5-fluorocytosine prodrug (P < 0.0001). 5-Fluorocytosine treatment of 4TCD(y), but not 4T1 cells, enhanced the effects of radiation in vitro (P < 0.0001). 5-Fluorocytosine prodrug treatment also increased the therapeutic potential of radiation in vivo. Mice with 4TCD(y) intrafemoral tumors showed increased effectiveness of radiation based on improved reductions in tumor size, reductions in tumorigenic osteolysis, and a decrease in skeletal fractures (P < 0.01).